Antibacterial evaluation of structurally amphipathic, membrane active small cationic peptidomimetics: Synthesized by incorporating 3-amino benzoic acid as peptidomimetic element

Article abstract of DOI:10.1016/j.ejmech.2014.06.023

A new series of small cationic peptidomimetics were synthesized by incorporating 3-amino benzoic acid (3-ABA) in a small structural framework with the objective to mimic essential properties of natural antimicrobial peptides (AMPs). The new design approac

Full text of DOI:10.1016/j.ejmech.2014.06.023

Accepted Manuscript
Antibacterial evaluation of structurally amphipathic, membrane active small
cationic peptidomimetics: Synthesized by incorporating 3-amino benzoic acid as
peptidomimetic element
Sandeep Lohan, Swaranjit Singh Cameotra, Gopal Singh Bisht
PII:
S0223-5234(14)00542-X
10.1016/j.ejmech.2014.06.023
EJMECH 7066
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 January 2014
Revised Date: 6 June 2014
Accepted Date: 12 June 2014
Please cite this article as: S. Lohan, S.S. Cameotra, G.S. Bisht, Antibacterial evaluation of structurally
amphipathic, membrane active small cationic peptidomimetics: Synthesized by incorporating 3-amino
benzoic acid as peptidomimetic element, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.06.023.
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*Graphical Abstract (for review)
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*Highlights (for review)
ACCEPTED MANUSCRIPT
Highlights



Synthesis of a series of 3-ABA based short cationic peptidomimetics.
Lead peptidomimetics showed activity against MRSA & MRSE.
Calcein dye leakage and fluorescent microscopy results display membrane disruption
action on intact bacterial cells.

Lead peptidomimetics were stable in plasma and even resistant pathogen (MRSA) was not
able to develop resistance against them.

*Revised Manuscript
Click here to view linked References
Antibacterial evaluation of As t rCu Cc tEu Pr aT l lEy Da mM p Ah iNp aU t Sh iCc ,R mI Pe Tm brane active small cationic
peptidomimetics: Synthesized by incorporating 3-amino benzoic acid as peptidomimetic
element
a
b
a
Sandeep Lohan , Swaranjit Singh Cameotra , Gopal Singh Bisht *
a
Department of Pharmacy, Jaypee University of Information Technology, Solan 173234,
India.
b
Environmental Biotechnology & Microbial Biochemistry, Institute of Microbial Technology,
Chandigarh 160036, India.
*
Corresponding Author: Dr. Gopal Singh Bisht
Department of Pharmacy, Jaypee University of Information Technology, Solan, India.
Email: bishtchem2005@gmail.com
Tel: 91-1792-239336
Fax: +91-1792-245362
1

ACCEPTED MANUSCRIPT
Abstract
A new series of small cationic peptidomimetics were synthesized by incorporating 3-amino
benzoic acid (3-ABA) in a small structural framework with the objective to mimic essential
properties of natural antimicrobial peptides (AMPs). The new design approach resulted into
improvement of activity and selectivity in comparison to linear peptides and allowed us to
better understand the influence of structural amphipathicity on biological activity. Lead
peptidomimetics displayed antibacterial activities against resistant pathogens (MRSA &
MRSE). A calcein dye leakage experiment revealed a membranolytic effect of 4g and 4l
which was further confirmed by fluorescence microscopy. In addition, proteolytic stability
and no sign of resistance development against S. aureus and MRSA demonstrate their
potential for further development as novel antimicrobial therapeutics.
Keywords: Antibacterial peptidomimetics, 3-amino benzoic acid, Antibiotic resistance,
Plasma stability.
2

ACCEPTED MANUSCRIPT
1
. Introduction
The near-explosive increased occurence in multi-drug resistant pathogens is recognized as a
severe menace to public health in nations around the globe. Moreover, nowadays treatment
options are becoming limited by the emergence of infections caused by pathogenic bacterial
strains that are resistant to conventional antibiotics viz methicillin-resistant Staphylococcus
aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-
resistant enterococci (VRE). Earlier, infections caused by multi-drug resistant bacterial
strains were mostly limited to the nosocomial environment but community acquired resistant
strains are now rising in prevalence [1-3]. There is an urgent need to develop classes of
antibiotics with different mechanisms of action in comparison to existing antibiotics.
In the last decade, a number of studies were conducted to explore the potential of small
cationic antimicrobial peptides (SCAMPs), designed by mimicking essential structural
features of AMPs, as future antibiotics, especially because of their enzymatic stability and
minimal propensity for resistance development [4-8]. AMPs are widespread in nature and
have been isolated from virtually all multi-cellular organisms ranging from insects to
humans. It has been well documented that these native peptide templates are vital
components of innate immunity, as all forms of life use them as weapons to ward off
pathogenic microbes in order to survive and thrive on this planet [9,10]. Although there is
great structural diversity among AMPs, some common structural features which might be
responsible for activity have been identified [11-13]. Structurally, native AMPs are quite
large molecules ranging from 12 to 50 amino acid residues and adopt an amphipathic
conformation in close proximity of biological membranes in such a way that hydrophobic and
hydrophilic moieties segregates on opposite face of the molecular framework [14]. At
physiological pH they usually possess a net positive charge (generally +2 to +9) due to the
abundance of basic amino acids (lysine and arginine) in their sequence [15,16]. The cationic
nature of these genes coded peptides drives them towards the negatively charged components
3

of bacterial cytoplasmic mem bA r aC n Ce Et hPa Tt Ep r Do v Mi d eAs Na nU i Sn iCt i Ra lI Pm To de of interaction. Further, the
hydrophobic interactions between bulky side chains of AMPs and acyl tails of membrane
phospholipids allow the peptides to penetrate the cell membrane [17,18]. The precise
mechanism for lytic activity of AMPs is still a matter of debate, though several mechanisms
have been proposed to describe the lipid-peptide interactions [19-23]. In addition, a number
of AMPs are frequently referred as “host defense peptides” and have been found to initiate an
adaptive immune system [10,24]. Apparently, AMPs with their non-specific modes of action
might indeed decrease pathogens ability to develop resistance. Further, AMPs possess a
broad antimicrobial activity spectrum against microbial strains resistant to the conventional
antibiotics [9,25,26]. Thus, without any doubt, these attractive therapeutic features associated
with AMPs underlines their candidature to develop them as today’s antibiotics.
In an attempt to search novel antibacterial peptidomimetics, here we describe a novel series
of small cationic peptidomimetics designed by incorporating 3-ABA in peptide sequences
composed of 2 to 5 amino acid residues. As it is well known fact that the structural features
of AMPs like the net content of charge and/or hydrophobic bulk and facial amphipathicity
play a crucial role in their biological activity [12,13,27]. Consequently, to explore the
structure-activity relationship of novel peptidomimetic templates we carried out the synthesis
by varying sequence as well as a number of both cationic and hydrophobic amino acid
residues. All peptidomimetic molecules were screened against Gram-positive and Gram-
negative bacterial strains including MRSA and MRSE. In vitro toxicity has been assessed by
measuring the lytic activity against human red blood cells. The bactericidal kinetic study of
lead peptidomimetics was also performed. The interaction with bacterial membrane mimic
liposomes has been used for an initial investigation of the bactericidal mechanism. The
membrane perturbation effect of lead peptidomimetic molecules was further confirmed by
fluorescent microscopy assay. To further assess the prospective of lead peptidomimetics as a
4

novel class of antibiotics, the Ap rCo Ct e Eo l Py tTi cE sDt a bMi l Ai t yN aUn dS Cp oR t eI Pn tTi al of pathogen (susceptible as
well as methicillin resistant S. aureus) to develop resistance against them was determined.
2
. Results and discussion
2
.1 Design and synthesis
Native AMPs and synthetically designed peptidomimetic templates comprised of genetically
coded amino acid residues can be susceptible to proteolytic degradation [3-7]. Backbone
modification is one of the synthetic approaches used to design peptidase immune
peptidomimetics [6,7]. In addition, it is well documented that biological activity of native
AMPs is depending on their secondary structure as well as net content of cationic charge and
hydrophobicity [9,10]. Therefore, incorporation of any suitable moiety in the peptide
backbone which provides a specific turn might improve stability and antimicrobial potential.
The aromatic γ-amino acid 3-ABA has a constrained structural framework due to the
presence of two principal dihedral angles θ1 (C2-C3) and θ2 (C1-C2), sandwiched between φ
o
(
N-C3) and ψ (C1-CO). They have a fixed dihedral angle of about 180 . Possibly, it may
attribute to its β-sheet like structure (Figure 1) [28]. Rao et al. have incorporated 3-ABA in
β-hairpin loop as turn inducing motif and shown that it result into very stable secondary
structures [29]. Lundy et al. reported synthetic analogues of α-defensin (HNP-1) synthesized
by incorporating constrained aromatic amino benzoic acids as β-turn motif exhibit broad-
spectrum antibacterial activity [30]. Recently, Lengyel et al. reported that incorporation of 3-
ABA in peptide sequence improved the stability of folded conformation in aqueous medium
without effecting the arrangement of side chain residues [31]. Based on these observations,
we hypothesize that the incorporation of constrained aromatic amino acid (3-ABA) could
mimic essential structural features of AMPs in such a way that optimum therapeutic index
may possibly be achieved.
Initially, a library of small cationic peptidomimetics (1a, 2a-2c, 3a-3e, 4a-4g) was
synthesized by incorporating 3-amino benzoic acid (3-ABA) as peptidomimetic element
5

(
Scheme 1). Arginine (Arg) a nAd CT Cr yEp Pt o Tp Eh aDn M( T rAp )N aUm Si nCo Ra Ic Pi dT residues were used to provide
cationic charge and hydrophobic bulkiness respectively. We preferred arginine (Arg) over
lysine because it behaves like charged moiety under all physiological conditions and also
guanidine group would exhibit higher electrostatic interaction with the negatively charged
phospholipids of bacterial cell membrane [8,32]. The selection of Trp was based on the
presumption that the indole nucleus would be inserted into the membrane with the
hydrophobic part interacting with the hydrophobic portion of the bilayer and the amine
function interacting more closely with the polar head-groups of the membrane [33]. Strom et
al. have previously documented that a specific content of both cationic charge and lipophilic
bulk is required to design potent synthetic mimics of AMPs [8]. Consequently,
peptidomimetic molecules were synthesized by varying the sequence of charged (Arg) and
hydrophobic (Trp) amino acid residues on both sides of 3-ABA. Further peptidomimetic
molecules (3f and 4h-4l) were synthesized by replacing Arg with unnatural cationic amino
acid ornithine (Orn) in the most active and moderately active molecules (3e and 4c-4g).
Structural modification was done by the substitution bulky hydrophobic residue Trp with Phe
(
4m-4p) among the most active molecules (4f, 4g, 4k and 4l). Finally, for comparative
analysis, we synthesized linear peptide sequences (5a-5d) without incorporating 3-ABA.
.2 Antibacterial activity
2
The activity results showed that peptidomimetic molecules were in general more potent
against S. aureus and their resistant strains (MRSA & MRSE) than other bacterial strains (B.
subtilis, E. coli, and P. aeruginosa) used in the study. These outcomes point out their
predominant anti-staphylococcal behavior which is in agreement with previous reports [4,8].
Peptidomimetic molecules 1a, 2a-2c, 3a-3d composed of 3, 4, and 5 amino acid residues,
showed virtually no antibacterial activity. In some cases moderate activity was observed with
the exception of the molecule 3e that exhibited MIC values of 12.5, 10, and 12.5 µg/mL
against S. aureus, MRSA, and MRSE respectively. The peptidomimetic molecule 3e was also
6

active against E. coli and P. a Ae r Cu gC i nE oPs Ta ,Ea Dl th Mo u Ag hN t Uh eS MC RI CI Pv Ta lues were considerably higher
in comparison to their activity against various strains of S. aureus (Table 1). Peptidomimetic
molecules (4a-4g) with 6 amino acid residues displayed higher antibacterial activity against
both Gram-positive and Gram-negative bacteria in comparison to peptidomimetic sequences
with tri, tetra and penta amino acid residues. Most active peptidomimetic molecule 4g
displayed maximum potency with MIC values in the range of 5-5.5 µg/mL against S. aureus,
MRSA, and MRSE. It was interesting to note that 4g showed comparable activity against
Gram-negative bacteria also with MIC values of 7.5 and 12.5 µg/mL against E. coli and P.
aeruginosa respectively. Moreover, peptidomimetic molecule 4f with 6 amino acid residues
was found to be active against Gram-positive as well as Gram-negative bacteria. Same
pattern of activity was observed in the case of 4l which exhibited MIC values in the range of
5
-6.25 µg/mL against S. aureus, MRSA, and MRSE. Peptidomimetic molecule 4l was found
to have somewhat low activity against Gram-negative bacteria with MICs 20 and 15.5 µg/mL
against E. coli and P. aeruginosa respectively. The peptidomimetic molecules having
phenylalanine as hydrophobic residue (4m-4p) showed insignificant lytic action against all
screened bacterial strains. It was interesting to note that the linear peptide sequences without
3
-ABA (5a-5d) showed poor antibacterial activity.
The activity results revealed a strong correlation between the antibacterial potency and
overall structural amphipathicity of peptidomimetics. This can be illustrated by observing
highest antibacterial activity in case of 4g and 4l which are having amphipathic structure. 3D
view of the structural framework of lead peptidomimetics (4g and 4l) and linear peptides (5b
and 5d) is shown in Figure 2. This structure-activity relationship was further confirmed by
observing bacterial killing effect of 3e, the only penta peptidomimetic having amphipathic
structural framework. The presence of a hydrophobic structural motif composed of 3-ABA
flanked by Trp residues is another common structural feature of active peptidomimetic
molecules (3e, 4f, 4g, 4k, and 4l; Figure 3). Wessolswski et al. have already reported that
7

small Arg-Trp rich AMPs w iAt hCt Ch rEe eP Ta dE j Da c eMn tAa Nr o Um Sa tCi cRr Ie Ps iTd ues display high bactericidal
action [34]. In this way this could be another reason for the efficient antibacterial potency of
newly designed peptidomimetics.
It was intresting to note that peptidomimetics 4f and 4g synthesized by incorporating 3-ABA
displayed 3-4 fold of improvements in activity against S. aureus, MRSA, and MRSE in
comparison to linear peptides 5a and 5b. In similar fashion, peptidomimetics having Orn as
cationic residue (4k and 4l) required nearly 8-10 fold of less concentration in comparison to
linear peptides (5c and 5d) to completely eradicate the growth of S. aureus, MRSA, and
MRSE (Table 1). Therefore, the results demonstrated that this novel design principle may
facilitate the development of new potent antibacterial peptidomimetics.
2
.3 Hemolytic activity
We performed hemolytic assay by quantifying 50% (HC ) as well as 10% hemolysis (HC )
5
0
10
against human red blood cells (hRBCs) as a measurement of toxicity. In general, lengthy
peptidomimetic derivatives (4a-4p) were found more hemolytic than smaller peptidomimetic
derivatives (1a, 2a-2c, and 3a-3f) as summarized in Table 1. Thus, it seems that, as the
hydrophobic bulk of peptidomimetic derivatives increases, their ability to discriminate
between anionic bacterial surface and zwitterionic mammalian membrane decreases. These
outcomes were in accordance with Kondejewski et al., who explored the correlation between
hydrophobicity and hemolytic activity [35]. Maximum hemolysis determined by HC was
1
0
detected for 4g at 80 µg/mL. Other peptidomimetic sequences caused 10% hemolysis at a
concentration above 100 µg/mL. Therefore, overall outcomes of hemolytic assay revealed
that all synthesized peptidomimetics do not readily induce lysis of hRBCs. Furthermore,
selectivity ratio (SR) was calculated by dividing HC50 values with the MICs against S.
aureus. The maximum selectivity was observed for one of the lead peptidomimetic molecule
4
l (SR= 62.4) followed by 4g (SR= 62; Table 1). In addition, the hemolytic activity results
showed that our structural model increased the selectivity of peptidomimetic molecules as
8

lead peptidomimetics with 3 -AA CB AC Es Pc aT f Ef o Dl d M( 4 Af , N4 Ug ,S 4Ck R, Ia Pn dT 4l) displayed low hemolytic
activity in comparison to linear peptides (5a-5d) as summarized in Table 1.
2
.4. Cytotoxicity
The cytotoxicity of lead peptidomimetics (4g & 4l) and linear peptides (5b & 5d) was
assessed by using MTT and LDH assays. The results of MTT assay showed that tested
molecules (4g, 4l, 5b, and 5d) caused a decrease in cell viability in a concentration
dependent manner (Figure 4). However, 5b and 5d were found to be comparatively higher
toxic toward HaCaT cells. Similarly, LDH assay results further revealed the lower toxicity of
lead peptidomimetics (4g & 4l) in comparison to linear peptides (5b & 5d) (Figure 5). At
each concentration level 5b and 5d induced higher levels of LDH release when compared
with 4g and 4l.
2
.5. Bactericidal kinetic assay
In comparison to the conventional antibiotics, AMPs are having fast bacterial killing kinetics
26,36]. To determine whether this ability is also inherent to newly designed peptidomimetics
[
reported here, the viability of exponentially growing S. aureus and E. coli was checked
against most potent peptidomimetics (4g and 4l) by time-kill assay. Bactericidal kinetic study
results demonstrated the rapid killing effect of lead peptidomimetics. Compound 4g and 4l at
6
3
4
× MIC decrease the number of S. aureus and E. coli from 10 CFU/mL to nearly10
CFU/mL with in 30 min (Figure 6A and 6B). The rapid bactericidal effect is usually observed
among most of the membrane active AMPs. Thus, rapid bacterial killing effect of lead
peptidomimetics (4g and 4l) suggests that their antibacterial action might be mediated
through permeabilization of the bacterial membrane.
2
.6. Calcein dye leakage
Membrane disruption is generally considered as the plausible mode of action for most of the
naturally occurring AMPs and synthetically designed small cationic antimicrobial
peptidomimetics [25,37,38]. In order to assess membrane interaction behavior of 4g and 4l,
9

we performed calcein dye Al e Ca kCa gE eP Te xE pDe r iMm Ae n Nt Ub Sy C dRe sI iPg Tn ing negatively charged and
zwitterionic calcein encapsulated liposomes which mimics the outer surface of bacterial and
mammalian cell membranes respectively. Fluorescent dye leakage experiment results showed
that 4g induces dye leakage in a concentration dependent manner, as 47% of dye was released
at a highest experimental concentration (15 µg/mL) after approx. 20 min and 19% dye was
released at lowest concentration (5 µg/mL) after nearly 50 min. In an analogous manner to
4
g, 4l also induces dye leakage in a dose dependent manner as 23% leakage was observed
after 90 min at 5 µg/mL that increased to 48% at a concentration of 15 µg/mL after the same
time interval (Figure 7). While in case of mammalian membrane mimic liposomes negligible
amount of dye leakage was observed even at maximum used experimental concentration (15
µg/mL) of both 4g and 4l. Therefore, results of dye leakage experiment support the activity
results as 4g exhibits higher MIC values against all tested strains of bacteria in comparison to
4
l. The calcein dye leakage experiment indicated that, the biological activity of these
peptidomimetics is dependent on their ability to permeate target membranes, same as that of
AMPs.
2
.7. Fluorescence Microscopy
To further investigate the mechanism involved in antibacterial activity of newly designed
peptidomimetics, the ability of two most potent sequences 4g and 4l to cause membrane
damage was assessed by fluorescence microscopy (Figure 8). The effect of active sequences
on both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria were studied by a
double staining method with DAPI (4’,6-diamidino-2-phenylindole) and PI (propidium
iodide). In the first step bacterial strains (E. coli and S. aureus) were incubated with 4g and 4l
for 2 h after that cells were stained with DAPI, which stains all bacterial cells in blue
irrespective of their viability and PI, which penetrates only injured or dead cells with
compromised membranes. In control (without initial treatment with peptidomimetics), both
E. coli and S. aureus showed blue fluorescence with DAPI, while negligible number of cells
1
0

had red fluorescence with PI. OA nC t Ch eE oP t hT eEr Dh a nMd ,A i nN cUa sS eCo Rf It rPe Ta ted E. coli and S. aureus cells,
stronger red fluorescence was observed with PI (Figure 8). Comparatively intense red
fluorescence was observed for S. aureus, suggesting their significant membrane damage,
which fully supports our activity results. These results collectively indicate that 4g and 4I
effectively arrest bacterial growth, via a membrane disruption mechanism similar to most of
the native AMPs.
2
.8. Resistance study
The efficacy of conventionally used antibiotics in treating infections caused by drug resistant
pathogens has been diminished as a result of pathogens ability to switch on to an alternate
metabolic pathway. It has already been reported that low propensity of resistance
development was observed in case of therapeutics that kill bacteria by targeting cell
membrane [25,39]. Therefore, we have determined the potential of susceptible pathogenic S.
aureus as well as MRSA, a clinically relevant drug resistant bacteria pathogen, to develop
drug resistance against most potent sequences 4g and 4l. The new MIC values were
determined every 24 h after propagation of bacterial cultures with fresh media and serially
diluted concentrations of tested peptidomimetics. To make comparative analysis, parallel
cultures were exposed to 2-fold dilutions of the antibiotic ciprofloxacin as a positive control.
The experiment was repeated for 16 days. Results in figure 9 show the low propensity of
bacterial pathogens to develop resistance against 4g and 4l as there was almost no change in
the MIC after 16 passages.
2
.9. Plasma stability study
Most of the naturally occurring AMPs are fairly large molecules, and therefore are having
number of scissile amide bonds in their structural framework that can be easily targeted by
proteolytic enzymes [6,7]. The inherent instability of these potent antimicrobial templates
renders them considerably inactive in intended bioenvironment. Therefore, this is an issue
that needs to be resolved for commercialization of these promising compounds. In this
1
1

respect, to assess the biocomp Aa t iCb iCl i Et yP oTf El eDa d Mp eAp tNi d Uo mS Ci mRe It iPc Ts 4g and 4l, we conducted their
stability study in human blood plasma. 4g was not degraded when incubated in plasma for 1 h
and minute degradation (6.58%) was observed after 2 h incubation in plasma. It was
interesting to note that further degradation of 4g did not take place even after 24 h incubation
in plasma as no extra peak was observed (see Supporting Information; Figure S1). On the
other hand, no sign of degradation was detected for 4l even after 24 h incubation in plasma
(Figure S2). This is may be due to the presence of non genitically coded amino acid Orn,
which is not a well known substrate for proteolytic enzymes.
To determine the effectiveness of our novel synthetic approach regarding proteolytic stability
we also carried out the plasma stability study of linear peptides (5b & 5d). Minute
degradation (5.31%) of 5b was detected after 30 min incubation and it was further increased
(11.79%) when incubated for 1 h in plasma. No more enzymatic degradation of 5b was takes
place when incubated in plasma for 12 h and 18% of 5b was degraded when incubated in
plasma for 24 h (Figure S3). Small amount of enzymatic degradation (4.72%) was detected
for 5d after 30 min incubation in plasma and it was no more degraded when incubated in
plasma for 24 h (Figure S4). The outcomes of stability study revealed that incorporation of 3-
ABA enhanced the stability against proteolytic enzymes as 4g and 4l were found to be higher
stable in plasma in comparison to linear peptides 5b and 5d.
2
.10. Proteolytic stability
To further ensure the proteolytic stability of lead peptidomimetics, we carried out trypsin and
α-chemotrypsin digestion assay. The results from the stability study on peptidomimetics (4g
and 4l) and linear peptides (5b and 5d) were rather striking. 4g was found stable against
trypsin when incubated for 36 h and after that it was slowly degraded with approximately
6
5% of the peptidomimetic remaining after 4 days (Figure 10A). However, linear peptide
sequence (5b) showed higher susceptibility towards trypsin as only 21% of the peptide
remaining after 4 days. Noticeably, a negligible amount of tryptic degradation was observed
1
2

for 4l. In addition, linear pepti dA eC5 Cd Ea lPs oT Es hDo wMe dA gNo oU dSs Ct a Rb iIl Pi t Ty with nearly 82% of the parent
peptide remaning after 4 days. It is well documented that trypsin cleaves C-terminal to native
cationic amino acids such as Lys and Arg [6]. Thus, the presence of non-natural cationic
amino acid Orn might be responsible for the high stability of 4l and 5d against tryptic
degradation. Furthermore, the high stability of 4g and 4l as compared to 5b and 5d
respectively, showed that 3-ABA may be a valuable building block by protecting scissile
peptide bonds against tryptic degradation. Peptidomimetics 4g and 4l were seemingly
completely stable against proteolytic degradation by α-chemotrypsin for 24 h, and after that
slow degradation was observed with nearly 70% of the peptidomimetics were still intact after
4
days (Figure 10B). On the other hand, linear peptides (5b and 5d) were gradually degraded
when incubated with α-chemotrypsin and only 44% of the intact peptides remaining after 4
days. Mechanistically, α-chymotrypsin cleaves the peptide bond on the C-terminal side of
bulky hydrophobic amino acids (Phe, Trp) [7]. Accordingly, the better stability of 4g and 4l
against α-chymotrypsin might be due to the presence of 3-ABA. The results, thereby
collectively demonstrated that the incorporation of Orn as well as 3-ABA could render the
peptide sequences immune against proteolytic enzymes.
3
. Conclusions
In conclusion, our novel structural model has resulted in the identification of potent
antibacterial peptidomimetics that exhibit higher activity as compared to peptide sequences
without 3-ABA. In addition, newly designed peptidomimetic templates show specificity
toward bacteria relative to eukaryotic red blood cells. Based on the results of calcein leakage
experiment and fluorescent microscopic data on lead peptidomimetics 4g and 4l, we propose
that they are more prone to membrane damage mode of action which is similar to most of the
native AMPs. The lead compounds 4g and 4l display other essential properties such as
improved proteolytic stability and insignificant capability of pathogens to develop resistance
1
3

against them. Thus, the lead o Ab t Ca i Cn eEd Pf Tr oEm Dp Mr e sAe nNt Us t Su dC yRc Io Pu Tl d be optimized for therapeutic
use against drug resistant bacterial strains.
4
. Experimental Section
4
.1. Chemicals and reagents
Rink amide MBHA resin and protected amino acids Fmoc-Arg(pbf)-OH, Fmoc-Trp(Boc)-
OH, Fmoc-Phe-OH and Fmoc-Orn(Boc)-OH were purchased from Novabiochem (Mumbai,
India). Aromatic amino acid 3-amino benzoic acid and Fmoc-Cl were obtained from
Spectrochem (Mumbai, India). Other reagents used for solid phase synthesis of
peptidomimetic
molecules
included
N-hydroxybenzotrizole
(HOBt),
N,N’-
diisopropylcarbodiimide (DIC), Piperidine, N,N-dimethylformamide (DMF) (Spectrochem,
Mumbai, India), dimethylsulphoxide (DMSO), dichloromethane (DCM), 1,2-Ethanedithiol
(Merck, Mumbai, India), and Trifluoro acetic acid (TFA; Loba chemie, Mumbai, India).
Membrane bilayers mimic phospholipids dipalmitoylphosphatidylcholine (DPPC) and
dipalmitoylphosphatidylglycerol (DPPG) were purchased from Avanti Polar Lipids (New
Delhi, India). Calcein, propidium iodide, DAPI and buffer material were purchased from
Sigma-Aldrich (India). Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were obtained from Sigma
Chemical Co. (St Louis, MO, USA). The lactate dehydrogenase (LDH) assay kit was
purchased from Promega (Madison, WI, USA). All the solvents used for purification were of
HPLC grade and obtained from Merck (Mumbai, India). Buffers were prepared in double-
distilled water.
4
.2. General procedure for the Fmoc protection of 3-amino benzoic acid
To a solution of 3-amino benzoic acid (1.37 g, 10 mmol) in water (35 mL), was added
sodium hydrogen carbonate (2.52 g, 30 mmol), and the resulting mixture was cooled to 5 °C
and it was slowly added with Fmoc-Cl (3.87 g, 15 mmol) as a solution in para-dioxane. The
resulting mixture was stirred at 0 °C for 1 h and allowed to warm to room temperature
1
4

overnight. Completion of the Ar eCa Cc t Ei o Pn T wE aDs Mm oAn iNt o Ur e Sd C bRy I Pp Tr ecoated TLC plate. After the
completion of reaction water was added to the reaction mixture and the aqueous layer was
extracted with ethyl acetate. Then the organic layer was extracted twice with a saturated
aqueous solution of sodium bicarbonate. The combined aqueous layers were acidified to a
pH of 2 with 10% HCl, and then extracted three times with ethyl acetate. The combined
organic layers were removed under reduced pressure to isolate the title compound. The crude
material was used without any further purification.
1
4
.2.1. Fmoc-3-ABA: H NMR (400 MHz, DMSO d ) δ: 12.21 (s, 1H), 9.81 (s, 1H), 7.91 - 7.66
6
(
m, 6H), 7.52 - 7.27 (m, 6H), 4.47 - 4.45 (d, J = 8Hz, 2H), 4.26 - 4.23 (t, J = 12Hz, 1H).
MALDI-TOF: calcd for C H NO : 359.12, found 360.39.
22
17
4
4
.3. General procedure for solid phase synthesis of peptidomimetics
All peptidomimetics were synthesized manually following standard Fmoc solid phase
protocols using Rink amide-4-methylbenzhydrylamine hydrochloride salt (MBHA) resin
(loading 0.79 mmol/g) as solid support [40]. Rink amide resin (150 mg) was washed with
CH Cl (3 × 2 mL), which is followed by swelling in DMF (3.5 mL) for 25 min. The Fmoc
2
2
protecting group of resin was removed by treating with piperidine/DMF (20% v/v) mixture
for 10 min, followed by extensive washes with DMF (5 × 2 mL). The deprotection step was
performed twice. Each amino acid coupling step included an Fmoc deprotection and 3 h
coupling of 4 equiv of Fmoc protected amino acid onto resin in the presence 2 equiv of
DIC/HOBt in DMF. After the desired sequences were assembled, the peptidomimetic
molecules were cleaved with a solution of TFA/H O/1,2-Ethanedithiol (95:2.5:2.5) from
2
solid support (Scheme 1).
4
.4. Purification
All crude peptidomimetics were analysed on a reversed-phase high performance liquid
®
chromatography (RP-HPLC) using a C waters column (Spherisorb , ODS2, 5µm, 4.6 mm
1
8
×
250 mm) at room temperature. A linear gradient of 0.5-60% solvent B (0.05% TFA in
1
5

acetonitrile) in solvent A (0.0 5A% C TC F EA P iTn Ew Da t eMr ) Ao vN e Ur 3S 5C mR i In P, Tf ollowed by 60-0.5% solvent B
over 10 min was used at a flow rate of 0.5 mL/min. Preparative RP-HPLC was then
®
performed on a Waters column (Spherisorb , ODS2, 5µm, 20 mm × 250 mm) using 0.5-60%
linear gradient of solvent B (0.05% TFA in acetonitrile) in solvent A (0.05% TFA in water)
over 35 min, followed by 60-0.5% solvent B over 10 min at a flow rate of 5 mL/min. Mass
spectra were obtained on a Agilent MALDI-TOF mass spectrometer. Purified HPLC fractions
1
were than lyophilized. See the supporting information for analytical data (Mass spectra, H
NMR, and HPLC chromatograms).
1
4
2
7
.4.1. H-Trp-3ABA-Arg-NH (1a): H NMR (400 MHz, DMSO d ) δ: 10.98 (s, 1H), 10.91 (s,
2
6
H), 10. 80 (s, 1H), 10.76 (s, 2H), 9.42 (s, 2H), 8.80 (s, 1H), 8.16 - 8.15 (d, J = 4Hz, 2H),
.33 - 6.97 (m, 8H), 4.67 - 4.64 (t, J = 12Hz, 1H), 4.22 - 4.19 (t, J = 12Hz, 1H), 3.19 - 3.18
(d, J = 4Hz, 5H), 2.84 (s, 1H), 2.46 - 2.45 (t, J = 4Hz, 3H), 1.25 - 1.22 (m, 2H), 1.12 - 1.11
(
m, 2H). MALDI-TOF: calcd for C H N O : 478.24, found 479.24; Purity determined by
2
4
30
8
3
RP-HPLC: Retention time: 26.55 min, purity: 97.61%.
1
4
1
7
3
.4.2. H-Trp-Trp-3ABA-Arg-NH (2a): H NMR (400 MHz, DMSO d ) δ: 11.19 (s, 2H),
2
6
0.90 (s, 1H), 10.82 (s, 1H), 10. 76 (s, 2H), 10.34 (s, 2H), 9.37 (s, 1H), 8.63 (s, 2H), 7.90 -
.89 (d, J = 4Hz, 2H), 7.30 - 6.97 (m, 12H), 4.67 - 4.65 (m, 2H), 4.39 - 4.38 (t, J = 4Hz, 1H),
.19 - 3.18 (d, J = 4Hz, 5H), 2.84 (s, 1H), 2.47 - 2.45 (m, 3H), 1.25 - 1.11 (m, 4H). MALDI-
TOF: calcd for C H N O : 664.32, found 665.12; Purity determined by RP-HPLC:
3
5
40 10
4
Retention time: 28.66 min, purity: 98.62%.
1
4
1
7
3
.4.3. H-Arg-Trp-3ABA-Arg-NH (2b): H NMR (400 MHz, DMSO d ) δ: 11.12 (s, 1H),
2
6
0.86 (s, 2H), 10.80 (s, 2H), 10. 32 (s, 1H), 10.14 (s, 2H), 9.19 (s, 2H), 8.44 (s, 2H), 7.93 -
.91 (d, J = 8Hz, 2H), 7.31 - 7.01 (m, 7H), 4.62 - 4.57 (m, 2H), 4.37 - 4.35 (t, J = 8Hz, 1H),
.21 - 3.20 (d, J = 4Hz, 2H), 2.81 (s, 2H), 2.43 - 2.40 (m, 4H), 1.27 - 1.12 (m, 8H). MALDI-
TOF: calcd for C H N O : 634.35, found 635.44; Purity determined by RP-HPLC:
3
0
42 12
4
Retention time: 23.82 min, purity: 98.77%.
1
6

1
4
1
7
3
.4.4. H-Trp-Arg-3ABA-Arg-N AH C C( 2Ec P) :T EH DN MM AR N( 4U0 S0 C MR HI Pz ,T DMSO d ) δ: 11.09 (s, 1H),
2
6
0.81 (s, 2H), 10.79 (s, 2H), 10. 41 (s, 1H), 10.02 (s, 2H), 8.97 (s, 2H), 8.29 (s, 2H), 7.87 -
.85 (d, J = 8Hz, 2H), 7.35 - 7.07 (m, 7H), 4.71 - 4.66 (m, 2H), 4.32 - 4.31 (t, J = 4Hz, 1H),
.19 - 3.17 (d, J = 8Hz, 2H), 2.81 (s, 2H), 2.49 - 2.45 (m, 4H), 1.28 - 1.14 (m, 8H). MALDI-
TOF: calcd for C H N O : 634.35, found 635.39; Purity determined by RP-HPLC:
3
0
42 12
4
Retention time: 24.18 min, purity: 97.34%.
1
4
1
7
1
.4.5. H-Trp-Trp-3ABA-Arg-Arg-NH (3a): H NMR (400 MHz, DMSO d ) δ: 11.14 (s, 2H),
2 6
0.80 (s, 2H), 10.76 (s, 2H), 10. 29 (s, 2H), 9.91 (s, 4H), 8.90 (s, 2H), 8.22 (s, 2H), 7.74 -
.71 (d, J = 12Hz, 2H), 7.29 - 6.98 (m, 12H), 4.82 - 4.77 (m, 3H), 4.30 - 4.29 (t, J = 4Hz,
H), 3.18 - 3.16 (d, J = 8Hz, 4H), 2.80 (s, 2H), 2.51 - 2.46 (m, 4H), 1.29 - 1.13 (m, 8H).
MALDI-TOF: calcd for C H N O : 820.42, found 821.19; Purity determined by RP-
41
52 14
5
HPLC: Retention time: 25.37 min, purity: 95.66%.
1
4
1
7
3
.4.6. H-Arg-Trp-3ABA-Arg-Arg-NH (3b): H NMR (400 MHz, DMSO d ) δ: 11.04 (s, 1H),
2
6
0.78 (s, 6H), 10.71 (s, 2H), 10. 27 (s, 2H), 9.93 (s, 4H), 8.88 (s, 3H), 8.20 (s, 2H), 7.71 -
.69 (d, J = 8Hz, 2H), 7.28 - 7.09 (m, 8H), 4.80 - 4.74 (m, 3H), 4.29 - 4.27 (t, J = 8Hz, 1H),
.16 - 3.15 (d, J = 4Hz, 2H), 2.81 (s, 3H), 2.50 - 2.43 (m, 6H), 1.26 - 1.11 (m, 13H). MALDI-
TOF: calcd for C H N O : 790.45, found 791.44; Purity determined by RP-HPLC:
3
6
54 16
5
Retention time: 22.534 min, purity: 95.67%.
1
4
1
7
3
.4.7. H-Trp-Arg-3ABA-Arg-Arg-NH (3c): H NMR (400 MHz, DMSO d ) δ: 11.07 (s, 1H),
2 6
0.81 (s, 6H), 10.70 (s, 2H), 10. 29 (s, 2H), 9.71 (s, 4H), 8.81 (s, 3H), 8.25 (s, 2H), 7.71 -
.69 (d, J = 8Hz, 2H), 7.31 - 7.11 (m, 8H), 4.82 - 4.74 (m, 3H), 4.30 - 4.27 (t, J = 12Hz, 1H),
.18 - 3.16 (d, J = 8Hz, 2H), 2.83 (s, 3H), 2.51 - 2.46 (m, 6H), 1.21 - 1.10 (m, 13H). MALDI-
TOF: calcd for C H N O : 790.45, found 791.41; Purity determined by RP-HPLC:
3
6
54 16
5
Retention time: 22.640 min, purity: 98.38%.
1
4
.4.8. H-Trp-Arg-3ABA-Trp-Arg-NH (3d): H NMR (400 MHz, DMSO d ) δ: 11.10 (s, 2H),
2 6
1
0.87 (s, 2H), 10.70 (s, 2H), 10. 12 (s, 2H), 9.84 (s, 4H), 8.79 (s, 2H), 8.23 (s, 2H), 7.65 -
1
7

7
3
.63 (d, J = 8Hz, 2H), 7.36 - 7 A. 0 C2 C( mE , P1 T3 HE )D, 4 M. 7 9A -N 4 U. 7 S3 C( mR ,I P3 HT ), 4.29 - 4.27 (t, J = 8Hz, 1H),
.20 - 3.17 (d, J = 12Hz, 5H), 2.84 (s, 2H), 2.53 - 2.44 (m, 4H), 1.26 - 1.11 (m, 9H). MALDI-
TOF: calcd for C H N O : 820.42, found 821.33; Purity determined by RP-HPLC:
4
1
52 14
5
Retention time: 24.98 min, purity: 96.14%.
1
4
1
7
3
.4.9. H-Arg-Trp-3ABA-Trp-Arg-NH (3e): H NMR (400 MHz, DMSO d ) δ: 11.12 (s, 2H),
2 6
0.81 (s, 2H), 10.78 (s, 2H), 10. 23 (s, 2H), 9.86 (s, 4H), 8.94 (s, 2H), 8.19 (s, 2H), 7.72 -
.70 (d, J = 8Hz, 2H), 7.33 - 6.99 (m, 14H), 4.86 - 4.81 (m, 3H), 4.33 - 4.31 (t, J = 8Hz, 1H),
.20 - 3.18 (d, J = 8Hz, 4H), 2.84 (s, 2H), 2.56 - 2.49 (m, 4H), 1.27 - 1.13 (m, 8H). MALDI-
TOF: calcd for C H N O : 820.42, found 821.41; Purity determined by RP-HPLC:
4
1
52 14
5
Retention time: 23.89 min, purity: 98.17%.
1
4
1
7
4
1
.4.10. H-Trp-Trp-Arg-3ABA-Arg-Arg-NH (4a): H NMR (400 MHz, DMSO d ) δ: 10.96 (s,
2 6
H), 10.90 (s, 3H), 10.82 (s, 2H), 10. 76 (s, 4H), 10.35 (s, 2H), 9.37 (s, 6H), 8.51 (s, 2H),
.94 - 7.93 (d, J = 4Hz, 2H), 7.75 - 7.57 (m, 4H), 7.48 - 6.96 (m, 8H), 4.70 - 4.65 (m, 3H),
.21 - 4.19 (t, J = 8Hz, 2H), 3.14 - 3.12 (d, J = 8Hz, 8H), 2.83 (s, 3H), 2.47 - 2.45 (m, 6H),
.24 - 1.08 (m, 14H). MALDI-TOF: calcd for C H N O : 976.53, found 977.23; Purity
47
64 18
6
determined by RP-HPLC: Retention time: 27.340 min, purity: 98.42%.
1
4
1
7
4
1
.4.11. H-Arg-Trp-Arg-3ABA-Trp-Arg-NH (4b): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.88 (s, 3H), 10.80 (s, 2H), 10. 77 (s, 4H), 10.32 (s, 2H), 9.34 (s, 6H), 8.52 (s, 2H),
.95 - 7.94 (d, J = 4Hz, 2H), 7.71 - 7.53 (m, 4H), 7.44 - 6.90 (m, 8H), 4.68 - 4.61 (m, 3H),
.24 - 4.22 (t, J = 8Hz, 2H), 3.13 - 3.11 (d, J = 8Hz, 8H), 2.84 (s, 3H), 2.47 - 2.44 (m, 6H),
.25 - 1.07 (m, 14H). MALDI-TOF: calcd for C H N O : 976.53, found 977.29; Purity
47
64 18
6
determined by RP-HPLC: Retention time: 27.446 min, purity: 98.07%.
1
4
1
7
4
.4.12. H-Arg-Trp-Trp-3ABA-Arg-Arg-NH (4c): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.89 (s, 3H), 10.81 (s, 2H), 10. 76 (s, 4H), 10.36 (s, 2H), 9.39 (s, 6H), 8.52 (s, 2H),
.96 - 7.93 (d, J = 12Hz, 2H), 7.71 - 7.58 (m, 4H), 7.44 - 6.91 (m, 8H), 4.69 - 4.64 (m, 3H),
.22 - 4.20 (t, J = 8Hz, 2H), 3.13 - 3.11 (d, J = 8Hz, 8H), 2.82 (s, 3H), 2.46 - 2.43 (m, 6H),
1
8

1
.25 - 1.08 (m, 14H). MALD AI - CT OC FE :P cTa El c Dd fMo rACN UH S CN R IO P T: 976.53, found 977.19; Purity
47
64 18
6
determined by RP-HPLC: Retention time: 26.960 min, purity: 96.61%.
1
4
1
7
4
1
.4.13. H-Trp-Arg-Arg-3ABA-Trp-Arg-NH (4d): H NMR (400 MHz, DMSO d ) δ: 10.97 (s,
2
6
H), 10.89 (s, 3H), 10.82 (s, 2H), 10. 76 (s, 4H), 10.35 (s, 2H), 9.39 (s, 6H), 8.52 (s, 2H),
.95 - 7.93 (d, J = 8Hz, 2H), 7.72 - 7.54 (m, 4H), 7.40 - 6.90 (m, 8H), 4.70 - 4.64 (m, 3H),
.22 - 4.20 (t, J = 8Hz, 2H), 3.13 - 3.12 (d, J = 4Hz, 8H), 2.84 (s, 3H), 2.47 - 2.45 (m, 6H),
.24 - 1.08 (m, 14H). MALDI-TOF: calcd for C H N O : 976.53, found 977.27; Purity
47
64 18
6
determined by RP-HPLC: Retention time: 27.660 min, purity: 97.46%.
1
4
1
7
4
1
.4.14. H-Trp-Arg-Trp-3ABA-Arg-Arg-NH (4e): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.89 (s, 3H), 10.81 (s, 2H), 10. 76 (s, 4H), 10.36 (s, 2H), 9.42 (s, 6H), 8.55 (s, 2H),
.95 - 7.94 (d, J = 4Hz, 2H), 7.72 - 7.54 (m, 4H), 7.43 - 6.91 (m, 8H), 4.70 - 4.64 (m, 3H),
.22 - 4.20 (t, J = 8Hz, 2H), 3.13 - 3.11 (d, J = 8Hz, 8H), 2.84 (s, 3H), 2.48 - 2.45 (m, 6H),
.25 - 1.08 (m, 14H). MALDI-TOF: calcd for C H N O : 976.53, found 977.53; Purity
47
64 18
6
determined by RP-HPLC: Retention time: 27.324 min, purity: 96.06 %.
1
4
1
7
4
1
.4.15. H-Arg-Arg-Trp-3ABA-Trp-Arg-NH (4f): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.89 (s, 3H), 10.80 (s, 2H), 10. 76 (s, 4H), 10.36 (s, 2H), 9.39 (s, 6H), 8.52 (s, 2H),
.95 - 7.93 (d, J = 8Hz, 2H), 7.72 - 7.58 (m, 4H), 7.44 - 6.91 (m, 8H), 4.70 - 4.64 (m, 3H),
.22 - 4.19 (t, J = 12Hz, 2H), 3.13 - 3.12 (d, J = 4Hz, 8H), 2.84 (s, 3H), 2.47 - 2.45 (m, 6H),
.25 - 1.08 (m, 14H). MALDI-TOF: calcd for C H N O : 976.53, found 977.39; Purity
47
64 18
6
determined by RP-HPLC: Retention time: 27.402 min, purity: 99.65%.
1
4
3
8
4
1
.4.16. H-Trp-Arg-Trp-3ABA-Trp-Arg-NH (4g): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.91 (s, 2H), 10.80 (s, 1H), 10. 76 (s, 4H), 10.36 (s, 3H), 9.31 (s, 5H), 8.51 (s, 2H),
.16 - 8.15 (d, J = 8Hz, 2H), 7.74 - 7.56 (m, 8H), 7.44 - 6.84 (m, 9H), 4.70 - 4.62 (m, 2H),
.22 - 4.19 (t, J = 8Hz, 3H), 3.24 - 3.22 (d, J = 8Hz, 8H), 2.84 (s, 2H), 2.47 - 2.45 (m, 4H),
.25 - 1.22 (m, 4H), 1.13 - 1.07 (m, 4H). MALDI-TOF: calcd for C H N O : 1006.5,
5
2
62 16
6
found 1007.49; Purity determined by RP-HPLC: Retention time: 29.754 min, purity: 98.42%.
1
9

1
4
1
2
.4.17. H-Orn-Trp-3ABA-Trp- OA rCn C- N EH P T( 3E f D) : MH AN MN UR S( 4C0 R0 IMP HT z, DMSO d ) δ: 10.91 (s, 2H),
2
6
0.83 (s, 2H), 10.80 (s, 1H), 10. 21 (s, 2H), 9.50 (s, 3H), 8.54 (s, 4H), 8.14 - 8.12 (d, J = 8Hz,
H), 7.83 - 7.44 (m, 12H), 4.67 - 4.58 (m, 3H), 4.23 - 4.21 (t, J = 8Hz, 1H), 3.32 - 3.28 (d, J
=
16Hz, 5H), 2.49 - 2.44 (m, 5H), 1.72 - 1.61 (m, 4H), 1.26 - 1.08 (m, 4H). MALDI-TOF:
calcd for C H N O : 736.38, found 737.18; Purity determined by RP-HPLC: Retention
3
9
48 10
5
time: 24.231 min, purity: 98.74%.
.4.18. H-Orn-Trp-Trp-3ABA-Orn-Orn-NH (4h): H NMR (400 MHz, DMSO d ) δ: 10.97
1
4
2
6
(s, 2H), 10.85 (s, 2H), 10.78 (s, 2H), 10. 38 (s, 2H), 9.54 (s, 3H), 8.81 (s, 6H), 8.61 - 8.58 (d,
J = 12Hz, 2H), 8.51 - 8.42 (m, 4H), 8.35 - 7.88 (m, 4H), 7.49 - 6.99 (m, 5H), 4.70 - 4.61 (m,
3
2
H), 4.34 - 4.20 (m, 2H), 3.27 - 3.23 (d, J = 16Hz, 5H), 2.83 - 2.68 (m, 5H), 2.47 - 2.45 (m,
H), 1.72 - 1.55(m, 7H), 1.27 - 1.07 (m, 6H). MALDI-TOF: calcd for C H N O : 850.46,
4
4
58 12
6
found 851.47; Purity determined by RP-HPLC: Retention time: 23.979 min, purity: 96.31%.
1
4
2
1
4
1
8
4
2
1
4
1
8
4
.4.19. H-Trp-Orn-Orn-3ABA-Trp-Orn-NH (4i): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.84 (s, 1H), 10.81 (s, 2H), 10. 38 (s, 2H), 9.50 (s, 4H), 8.79 (s, 6H), 8.66 - 8.63 (d, J =
2Hz, 2H), 8.51 - 8.43 (m, 5H), 8.35 - 7.83 (m, 4H), 7.46 - 6.90 (m, 6H), 4.68 - 4.61 (m, 3H),
.37 - 4.24 (m, 2H), 3.52 - 3.47 (d, J = 20Hz, 5H), 2.83 - 266 (m, 6H), 2.47 - 2.45 (m, 2H),
.72 - 1.54 (m, 7H), 1.25 - 1.08 (m, 6H). MALDI-TOF: calcd for C H N O : 850.46, found
4
4
58 12
6
51.37; Purity determined by RP-HPLC: Retention time: 24.193 min, purity: 98.57%.
1
.4.20. H-Trp-Orn-Trp-3ABA-Orn-Orn-NH (4j): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.85 (s, 1H), 10.80 (s, 2H), 10. 41 (s, 2H), 9.52 (s, 4H), 8.79 (s, 6H), 8.64 - 8.61 (d, J =
2Hz, 2H), 8.47 - 8.39 (m, 5H), 8.33 - 7.80 (m, 4H), 7.45 - 6.88 (m, 6H), 4.70 - 4.58 (m, 3H),
.38 - 4.24 (m, 2H), 3.67 - 3.62 (d, J = 20Hz, 5H), 2.81 - 265 (m, 6H), 2.46 - 2.45 (m, 2H),
.71 - 1.54 (m, 7H), 1.26 - 1.07 (m, 6H). MALDI-TOF: calcd for C H N O : 850.46, found
4
4
58 12
6
51.41; Purity determined by RP-HPLC: Retention time: 24.782 min, purity: 98.38%.
1
.4.21. H-Orn-Orn-Trp-3ABA-Trp-Orn-NH (4k): H NMR (400 MHz, DMSO d ) δ: 10.98
2
6
(s, 2H), 10.85 (s, 1H), 10.81 (s, 1H), 10. 38 (s, 2H), 9.50 (s, 5H), 8.79 (s, 6H), 8.64 - 8.62 (d,
2
0

J = 8Hz, 2H), 8.49 - 8.42 (m, A5 HC )C, 8E . P3 4T E- 7D .8 M3 (Am N, 4U HS ) C, 7R. 4I 6P T- 6.90 (m, 6H), 4.69 - 4.61 (m,
3
2
H), 4.37 - 4.21 (m, 2H), 3.68 - 3.60 (d, J = 32Hz, 5H), 2.84 - 266 (m, 6H), 2.47 - 2.45 (m,
H), 1.72 - 1.54 (m, 7H), 1.25 - 1.07 (m, 6H). MALDI-TOF: calcd for C H N O : 850.46,
4
4
58 12
6
found 851.34; Purity determined by RP-HPLC: Retention time: 24.231 min, purity: 98.74%.
1
4
3
2
2
6
.4.22. H-Trp-Orn-Trp-3ABA-Trp-Orn-NH (4l): H NMR (400 MHz, DMSO d ) δ: 10.98 (s,
2
6
H), 10.87 (s, 1H), 10.82 (s, 1H), 10. 30 (s, 1H), 9.59 (s, 3H), 8.86 (s, 5H), 8.44 - 8.38 (d, J =
4Hz, 2H), 7.65 - 7.26 (m, 8H), 7.02 - 6.83 (m, 11H), 4.72 - 4.68 (m, 3H), 4.45 - 4.22 (m,
H), 3.21 - 3.17 (d, J = 16Hz, 6H), 2.84 - 264 (m, 5H), 2.47 - 2.45 (m, 2H), 1.71 - 1.54 (m,
H), 1.23 - 1.06 (m, 4H). MALDI-TOF: calcd for C H N O : 922.46, found 923.29; Purity
50
58 12
6
determined by RP-HPLC: Retention time: 25.378 min, purity: 99.66%.
1
4
.4.23. H-Arg-Arg-Phe-3ABA-Phe-Arg-NH (4m): H NMR (400 MHz, DMSO d ) δ: 10.90
2
6
(
s, 2H), 10.82 (s, 3H), 10. 77 (s, 3H), 10.35 (s, 6H), 9.39 (s, 2H), 8.51 (s, 2H), 7.58 - 7.56 (d,
J = 8Hz, 2H), 7.49 - 7.02 (m, 13H), 4.61 - 4.54 (m, 3H), 4.17 - 4.16 (t, J = 4Hz, 2H), 3.11 -
.10 (d, J = 4Hz, 6H), 2.83 (s, 3H), 2.47 - 2.45 (m, 6H), 1.24 - 1.07 (m, 13H). MALDI-TOF:
calcd for C H N O : 898.5, found 899.39; Purity determined by RP-HPLC: Retention time:
3
4
3
62 16
6
2
4.871 min, purity: 98.21%.
.4.24. H-Phe-Arg-Phe-3ABA-Phe-Arg-NH (4n): H NMR (400 MHz, DMSO d ) δ: 10.89
1
4
2
6
(
s, 2H), 10.80 (s, 3H), 10. 76 (s, 2H), 10.36 (s, 4H), 9.38 (s, 2H), 8.52 (s, 2H), 7.58 - 7.57 (d,
J = 4Hz, 2H), 7.52 - 7.21 (m, 11H), 7.14 - 6.98 (m, 8H), 4.60 - 4.54 (m, 3H), 4.17 - 4.15 (t, J
8Hz, 2H), 3.11 - 3.9 (d, J = 8Hz, 7H), 2.84 (s, 2H), 2.48 - 2.45 (m, 5H), 1.25 - 1.07 (m,
H). MALDI-TOF: calcd for C H N O : 889.47, found 890.33; Purity determined by RP-
=
9
4
6
59 13
6
HPLC: Retention time: 26.225 min, purity: 98.34%.
1
4
.4.25. H-Orn-Orn-Phe-3ABA-Phe-Orn-NH (4o): H NMR (400 MHz, DMSO d ) δ: 10.90
2 6
(s, 2H), 10.83 (s, 3H), 10.36 (s, 6H), 9.41 (s, 2H), 8.50 (s, 2H), 7.58 - 7.56 (d, J = 8Hz, 2H),
7
8
.50 - 7.02 (m, 14H), 4.61 - 4.54 (m, 3H), 4.17 - 4.15 (t, J = 8Hz, 2H), 3.12 - 3.10 (d, J =
Hz, 5H), 2.47 - 2.45 (m, 6H), 1.24 - 1.07 (m, 14H). MALDI-TOF: calcd for C H N O :
4
0
56 10
6
2
1

7
9
4
72.44, found 773.46; Purity Ad e Ct e Cr mE i Pn eT dE bDy MR PA- HN PUL SC C: RR eI tPe Tn tion time: 23.313 min, purity:
6.67%.
1
.4.26. H-Phe-Orn-Phe-3ABA-Phe-Orn-NH (4p): H NMR (400 MHz, DMSO d ) δ: 10.89
2
6
(s, 2H), 10.80 (s, 3H), 10.36 (s, 4H), 9.36 (s, 2H), 8.50 (s, 2H), 7.58 - 7.57 (d, J = 4Hz, 2H),
7
3
.53 - 7.20 (m, 10H), 7.16 - 6.98 (m, 8H), 4.60 - 4.54 (m, 3H), 4.17 - 4.15 (t, J = 8Hz, 2H),
.11 - 3.9 (d, J = 8Hz, 7H), 2.48 - 2.45 (m, 5H), 1.25 - 1.07 (m, 9H). MALDI-TOF: calcd for
C H N O : 805.43, found 806.36; Purity determined by RP-HPLC: Retention time: 25.038
44
55
9
6
min, purity: 98.28%.
.5. Antibacterial activity
4
The antibacterial activity of synthesized peptidomimetic molecules was determined by
screening against a range of susceptible as well as drug resistant strains includes Escherichia
coli (MTCC 723), Pseudomonas aeruginosa (MTCC 2295), Staphylococcus aureus (MTCC
3
160), Basilus subtilis (MTCC 2763), and methicillin-resistant bacterial strains MRSA
(ATCC BAA-1720) & MRSE (ATCC 51625). Drug resistant bacterial strains used in the
present study were collected from Medicos laboratories, Chandigarh, India. Antibacterial
susceptibility testing was carried out using a modification of the Clinical Laboratory Standard
Institute (CLSI) micro dilution broth assay [41]. The assay was performed in sterile 96-well,
flat-bottom polypropylene microtiter plates (SIGMA). Briefly, each well of 96-well plate was
5
inoculated with 90 μL of approximately 2 × 10 CFU/mL of bacterial suspension per mL of
Mueller-Hinton broth (HIMEDIA). Then 10 μL of serially diluted peptidomimetics over
concentration ranging from 0.7-100 μg/mL was added to the wells of microtiter plate. The
microtiter plates were incubated overnight with agitation at 37 °C and absorbance was read at
5
6
00 nm after 18 h. Cultures (approximately 2 × 10 CFU/ mL) without peptidomimetics were
used as positive control. Uninoculated Mueller-Hinton broth was used as negative control.
The tests were carried out in triplicate. The minimum inhibitory concentration (MIC) is
defined as the lowest concentration of peptide that completely inhibits growth.
2
2

4.6. Computational simulation ACCEPTED MANUSCRIPT
To analyze the dynamic behavior of the peptidomimetic molecules (4g, 4l, 5b, & 5d) we
performed molecular dynamic (MD) simulation by using UCSF CHIMERA version 1.4
(build 29530) on operating system win32. We carried out molecular mechanics operation in
aqueous environment. We have chosen the initial coordinates of the molecules and calculated
the energy by using conjugate gradient energy minimizer and perform a simulation. To
minimize the structure we predicted several starting points in energy landscape and the
optimal structure was one with lowest energy. Our experiment generated approximately 250
to 270 conformers for each molecule and after removal of duplicated minima; only
conformations within 3.2 kcal/mol of the global minimum were considered. Finally, we have
validated most stable structure for each molecule through Structure Analysis and Verification
Server (SAVS).
4
.7. Hemolytic assay
Hemolytic activity of the peptides was determined using fresh isolated hRBCs. The hRBCs
were centrifuged for 15 min to remove the buffy coat and washed three times with phosphate
buffer saline (35 mM phosphate buffer, 150 mM NaCl pH 7.2). 100 μL of the hRBC
suspended 4% (v/v) in PBS was plated into sterilized 96-well plates and then 100 μL peptide
solution (serial twofold dilution in PBS) was added to each well. The plates were incubated
for 1 h at 37 °C without agitation and centrifuged at 1000 g for 5 min. Aliquots (100 μL) of
the supernatant were transferred to 96-well plates, where hemoglobin release was monitored
using microtiter plate reader (Bio-Rad, India) by measuring the absorbance at 540 nm.
Percent hemolysis was calculated by the following formula:
%
age hemolysis = 100 × [(A - A )/(A - A )]
0
t
0
Where, A represents absorbance of peptide sample at 540 nm and A and A represent zero
0
t
percent and 100% hemolysis determined in PBS and 1% Triton X-100, respectively.
.8. Cell culture and cytotoxicity
4
2
3

HaCaT cells (Human keratino Ac y Ct e Cs ) EwP e Tr eE oDb t Ma i nAe dN fUr oSmC NR aI Pt i oT nal Centre for Cancer Science
NCCS), Pune and grown as a monolayer in DMEM supplemented with 10% FBS (Fetal
(
Bovine Serum), 100 µg/mL streptomycin and 100 units/mL penicillin. Cells were incubated
at 37 °C in an atmosphere of 5% CO2.
4
.8.1. MTT assay
The cell viability of HaCaT cells was assessed by the MTT colorimetric assay, which is based
on the reduction of MTT by the mitochondrial succinate dehydrogenase of intact cells to a
purple formazan product. Briefly, cells were incubated in 96-well microtiter plates for 24 h at
3
7 °C in a 5% CO incubator. Following the addition of the test peptidomimetics, the plates
2
were incubated for an additional 24 h. Control wells contained medium alone. Three replicate
wells were used at each point in the experiments. After 24 h incubation, MTT solution (5
mg/mL in phosphate-buffered saline) was added and incubated for another 4 h. The resulting
MTT/formazan product was dissolved by 100 µL of isopropanol and the plates were gently
shaken to solubilise the formed formazan. The amount of formazan was determined by
measuring the absorbance (OD) at 570 nm using a Bio-Rad 550 enzyme-linked
immunosorbent assay (ELISA) microplate reader [42].
Cell survival was calculated as the percentage MTT inhibition as follows:
%
growth inhibition = 100 − (mean OD of individual test group/ mean OD of each control
group) ×100.
.8.2. LDH assay
4
In LDH assay, leakage of the cytoplasmic located enzyme LDH into the extracellular medium
is measured. Briefly, cells were incubated in 96-well microtiter plates for 24 h at 37 °C in a
5
% CO incubator. At 24 h after cell seeding, cells were exposed to varying concentrations of
2
the test peptidomimetics. After 24 h of treatment, the supernatants were collected from each
well. Cell monolayers were trypsinized and the cells and lysate were collected. LDH activity
was measured in both the supernatants and the cell lysate fractions using Cyto-Tox 96, a non-
2
4

radioactive cytotoxicity assayA kCi t C, Ei nP aT c Ec oDr d Ma n Ac eN wU i tSh Ct Rh eI PmT anufacturer’s instruction. The
intensity of color is proportional to LDH activity. LDH release was calculated by measuring
the absorbance at 490 nm using ELISA microplate reader [43]. The percentage LDH release
from the cells was determined using the following formula:
%
LDH release = ((absorbance of the supernatant)/ absorbance of the supernatant and cell
lysate)) ×100
.9. Bactericidal kinetics
4
The time course of bacterial killing was studied by the exposure of S. aureus (MTCC 3160)
7
and E. coli (MTCC 723) cultures (2 × 10 CFU/mL) to lead peptidomimetics (4g and 4l) at 4
×
MIC in Muller Hinton media. Aliquots were removed at a fixed time interval, diluted up to
8
1
0 , plated on the Muller Hinton agar plate and CFU were counted after 24 h incubation at 37
°
C. Untreated bacterial culture was used as a control. Data were obtained from two
independent experiments performed in triplicate.
4
4
.10. Membrane interaction study using membrane models
.10.1. Preparation of calcein encapsulated liposomes
Large unilamellar vesicles (LUVs) of dipalmitoylphosphatidylcholine (DPPC) and
dipalmitoylphosphatidylglycerol (DPPG) were prepared to mimic bacterial membrane
(DPPC:DPPG, 7:3, w/w) or mammalian membrane (DPPC/ Chol, 10:1, w/w) as described
previously [44]. Briefly, a defined amount of lipid mixture DPPC:DPPG (7:3) was dissolved
in 2 mL chloroform/methanol solvent mixture in a 100 mL round bottom flask. The solvents
were removed under a stream of nitrogen and the lipid film obtained was lyophilized
overnight to remove any trace of solvent. The thin lipid film was rehydrated with calcein
containing buffer comprising 70 mM calcein, 150 mM NaCl, and 0.1 mM EDTA and
adjusted to pH 7.4 by the addition of a few drops of sodium hydroxide solution (1 M). The
liposome suspension obtained after rehydration was frozen and thawed for five cycles and
extruded 15 times through two stacked polycarbonate filters (100 nm pore size). The free
2
5

calcein was removed by passi An gCt Ch eE lPi pTo Es oDm Me s Au sNp eUn Ss i Co nR tI hP r To ugh a Sephadex G-50 column
and eluting with a buffer containing 10 mM Tris-HCl (150 mM NaCl, 0.1 mM EDTA). After
passing the liposome through Sephadex G-50, liposome diameter was measured by dynamic
light scattering using a Zetasizer Nano ZS (Malvern Instruments, India). Average diameter of
LUVs was found to be in the range of 110-130 nm.
4
.10.2. Calcein dye leakage assay
Peptidomimetic induced leakage of calcein from the LUVs was monitored by measuring the
fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of
5
20 nm on a Fluoromax 4 (spex) spectrofluorometer. Aliquots of liposome suspension were
then diluted in calcein free buffer (150 mM NaCl, 0.1 mM EDTA) to a final concentration of
0 μM lipid and incubated for 5 min with different concentrations of peptidomimetics (5-15
4
µg/mL). Calcein release from LUVs was assessed every minute for the first 20 min of the
experiment and after onward measurement was taken at the interval of 10 min. The
fluorescence intensity corresponding to 100% calcein release was determined by the addition
of a 10% solution (w/v) of Triton X-100. The apparent percentage of dye leakage was
calculated using the following formula:
%
Dye leakage = 100 × [(F - F )/ (F - F )]
0 t 0
Where F is the intensity measured at a given concentration of peptidomimetic, F is the
0
intensity of the liposomes (background), and F is the intensity after lysis by Triton X-100.
t
4
.11. Fluorescence microscopy
In order to shed light on bactericidal mechanism of newly designed peptidomimetics
fluorescence microscopy assay with DAPI (4’,6-Diamidino-2-phenylindole dihydrochloride,
Sigma) and PI (Propidium iodide, Sigma) as fluorophores was performed by treating E. coli
and S. aureus cells. In this double staining method we can easily visualize and differentiate
viable cells from the dead cells. DAPI as a double stranded DNA binding dye, stains all
bacterial cells irrespective of their viability. Whereas PI is capable of passing through only
2
6

damaged cell membranes and Ai n Ct e Cr c Ea lPa tTe Es Dw i tMh At h eN nU u Sc lCe iRc IaPc Ti ds of injured and dead cells to
form a bright red fluorescent complex. The cells were first stained with DAPI and then with
6
PI. Bacterial cells were grown until they reached mid-logarithmic phase (2 × 10 cells) and
then they were incubated with the peptidomimetics at the concentration of 4 × MIC for 2 h.
Then the cells were pelleted by centrifugation at 3000g for 15 min in an eppendorf
microcentrifuge. The supernatant was then decanted and the cells were washed with PBS
o
several times and then incubated with PI (5 μg/mL) in the dark for 15 min at 0 C. The excess
PI was removed by washing the cells with PBS several times. Then the cells were incubated
o
with DAPI (10 μg/mL) for 15 mins in dark at 0 C. The DAPI solution was removed and cells
were washed with PBS several times. Controls were performed following the exact same
procedure for bacteria without the treatment with peptidomimetics. The bacterial cells were
then examined by using the Nikon eclipse Ti microscope with an oil-immersion objective
(60×).
4
.12. Resistance study
We determine the potential of susceptible as well as methicillin resistant S. aureus to develop
antimicrobial resistance with our most potent peptidomimetic sequences 4g and 4l. The initial
MIC values of lead peptidomimetics (4g and 4l) and standard antibiotic ciprofloxacin against
both bacterial strains was obtained as described above. Serial passage and MICs
determination were performed in 96 well microtiter plate containing peptidomimetics, each
over a range of doubling dilution concentrations. After the incubation period 18 h the entire
content of the triplicate wells with a concentration of peptidomimetics permitting visible
6
growth were then used to prepare the bacterial dilution (approximately 2 × 10 CFU/mL) for
the successive exposure. The experiment was repeated for 16 days. As a positive control,
parallel cultures were exposed to two fold dilutions of the standard antibiotic (ciprofloxacin).
4
.13. Plasma stability study
2
7