Penetratin analogues acting as antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2010.10.025

The synthesis, in vitro evaluation, and conformational study of penetratin analogues acting as antifungal agents are reported. Different peptides structurally related with penetratin were evaluated. Analogues of penetratin rich in Arg, Lys and Trp amino acids were tested. In addition, HFRWRQIKIWFQNRRM[O]KWKK-NH₂, a synthetic 20 amino acid peptide was also evaluated. These penetratin analogues displayed antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. In contrast, Tat peptide, a well-known cell penetrating peptide, did not show a significant antifungal activity against fungus tested here. We also performed a conformational study by means experimental and theoretical approaches (CD spectroscopic measurements and MD simulations). The electronic structure analysis was carried out from Molecular Electrostatic Potentials (MEP) obtained by using RHF/6-31G ab initio calculations. Our experimental and theoretical results permitted us to identify a topographical template which may provide a guide for the design of new peptides with antifungal effects.

Full text of DOI:10.1016/j.ejmech.2010.10.025

European Journal of Medicinal Chemistry 46 (2011) 370e377
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Penetratin analogues acting as antifungal agents
,
,
,d
Francisco M. Garibotto a b, Adriana D. Garro a b, Ana M. Rodríguez a, Marcela Raimondi c
,
Susana A. Zacchino c, Andras Perczel e, Csaba Somlai f, Botond Penke f, Ricardo D. Enriz a
,b,
*
a Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina
b IMIBIO-SL, CONICET. Chacabuco 915, 5700 San Luis, Argentina
c Facultad de Ciencias Bioquímicas y Farmacéuticas, Farmacognosia, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina
d Facultad de Ciencias Médicas, Microbiología, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina
e HAS-ELU Protein Modelling Group, Institute of Chemistry, Eötvös L. University Pázmány P. s. 1/a Budapest, Hungary
f Department of Medical Chemistry, University of Szeged H-6720 Szeged, Dóm tér 8, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, in vitro evaluation, and conformational study of penetratin analogues acting as antifungal
agents are reported. Different peptides structurally related with penetratin were evaluated. Analogues of
penetratin rich in Arg, Lys and Trp amino acids were tested. In addition, HFRWRQIKIWFQNRRM[O]KWKK-
NH2, a synthetic 20 amino acid peptide was also evaluated. These penetratin analogues displayed anti-
fungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans.
In contrast, Tat peptide, a well-known cell penetrating peptide, did not show a significant antifungal
activity against fungus tested here. We also performed a conformational study by means experimental
and theoretical approaches (CD spectroscopic measurements and MD simulations). The electronic
structure analysis was carried out from Molecular Electrostatic Potentials (MEP) obtained by using RHF/6-
31G ab initio calculations. Our experimental and theoretical results permitted us to identify a topo-
graphical template which may provide a guide for the design of new peptides with antifungal effects.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 27 July 2010
Received in revised form
21 October 2010
Accepted 26 October 2010
Available online 29 October 2010
Keywords:
Antifungals
Candida albicans
Conformational study
Cryptococcus neoformans
Molecular electrostatic potentials
Peptides
1. Introduction
azole resistance among different pathogenic strains and the high
toxicity of amphotericin B [4,5] have prompted research for new
Many fungal infections are caused by opportunistic pathogens
that may be endogenous (Candida infections) or acquired from the
environment (Cryptococcus, Aspergillus infections). Patients with
significant immunosuppression frequently develop Candida
esophaguitis. Cryptococcosis, caused by the encapsulated yeast
Cryptococcus neoformans (C. neoformans), has been the cause of
fungal mortality among HIV-infected patients [1]. This organism has
predilection for the central nervous system and leads to severe, life-
threatening meningitis. Although it appears that many drugs are
available for the treatment of systemic and superficial mycoses,
there are in factonlya limited numberof efficacious antifungal drugs
[2]. Azoles that inhibit sterol formation and polyenes that bind to
mature membrane sterols have been the mainstays of antifungal
therapy for the last two decades [3]. However, the emergence of
antifungal agents [6]. Although combination therapy has emerged as
a good alternative to bypass these disadvantages [7,8], there is a real
need for a next generation of safer and more potent antifungal
agents [2,8]. This event resulted in the identification of novel
molecules, which might result useful for a future development.
Small cationic peptides [9,10] are abundant in nature and have
been described as ‘nature’s antibiotics’ or ‘cationic antimicrobial
peptides’. These peptides are 12e50 amino acids long with a net
positive charge of þ2 or þ9, due to an excess of basic arginine and
lysine, and with approximately 50% of hydrophobic amino acid
residues [9]. Penetratin, a well-known cell penetrating peptide
(CPP) and a synthetic 16 amino acid peptide from the third helix of
Antennapedia homeodomain [11], is a cationic amphipathic peptide
which might penetrate cell membranes via a postulated ‘inverted
micelle’ pathway [12]. This peptide has been proposed as
a universal intracellular delivery vehicle. Recently, we have repor-
ted that penetratin showed antifungal activity against Candida
albicans (C. albicans) and C. neoformans [13].
* Corresponding author. Facultad de Química, Bioquímica y Farmacia, Universidad
Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina. Tel.: þ54 2652
423789; fax: þ54 2652 431301.
In the present report, we extend our previous studies analyzing
different peptides structurally related to penetratin. Two principal
E-mail address: denriz@unsl.edu.ar (R.D. Enriz).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.025

F.M. Garibotto et al. / European Journal of Medicinal Chemistry 46 (2011) 370e377
371
goals were initially proposed for this work: i) to synthesize and test
2.3. Antifungal evaluation
new analogues of penetratin, and ii) to measure the antifungal
capacity of Tat peptide (another well-known CPP).
The test was performed in 96 wells-microplates. Peptide test
wells (PTW) were prepared with stock solutions of each peptide in
DMSO (ꢂ2%), diluted with RPMI-1640 to final concentrations
We also performed a conformational analysis from both experi-
mental (circular dichroism, CD) and theoretical methods (Electro-
statically Driven Monte Carlo (EDMC) calculations and Molecular
Dynamics (MD) simulations). Conformational and electronic anal-
yses were performed in order to confirm the pharmacophoric
pattern recently reported for these antifungal peptides [13,14].
200e3.125
mM. Inoculum suspension (100
ml) was added to each
well (final volume in the well ¼ 200
mL). A growth control well
(GCW) (containing medium, inoculum, the same amount of DMSO
used in PTW, but compound-free) and a sterility control well (SCW)
(sample, medium and sterile water instead of inoculum) were
included for each strain tested. Microtiter trays were incubated in
a moist, dark chamber at 35 ꢀC, 24 or 48 h for Candida spp or
Cryptococcus sp. respectively. Microplates were read in a VERSA
Max microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Amphotericin B (Sigma Chemical Co, St Louis, MO, USA) was used as
positive control (100% inhibition). Tests were performed by dupli-
cate. Reduction of growth for each peptide concentration was
calculated as follows: % of inhibition: 100- (OD405 PTW- OD405
SCW)/OD405 GCW - OD405 SCW.
2. Experimental
2.1. Synthetic methods
Solid phase synthesis of the peptides was carried out manually
on a p-methyl benzhydrylamine resin (1 g MBHA, 0.14 mmol/g)
with standard methodology using Boc-strategy. Side chain pro-
tecting groups were as follows: Arg(Tos), Lys(2Cl-Z), Cys(Mbzl), Tyr
(2-Br-Z). All protected amino acids were coupled in CH2Cl2 (5 ml)
using DCC (2.5 equiv.) and HOBt (2.5 equiv.) until completion (3 h)
judged by Kaiser [15] ninhydrin test. After coupling of the appro-
priate amino acid, Boc deprotection was effected by use of TFA/
CH2Cl2 (1:1, 5 ml)) for 5 min first then repeated for 25 min.
Following neutralization with 10% TEA/CH2Cl2 three times (5-5 ml
of each), the synthetic cycle was repeated to assemble the resin-
bond protected peptide. The peptides were cleaved from the resin
with simultaneous side chain deprotection by acidolysis with
anhydrous hydrogen fluoride (5 ml) containing 2% anisole, 8%
dimethyl sulfide and indole at 5 ꢀC for 45 min. The crude peptides
were dissolved in aqueous acetic acid and lyophilized. Preparative
and analytical HPLC of the crude and the purified peptides were
performed on an LKB Bromma apparatus (for preparative HPLC,
2.4. Statistical analysis
Data were statistical analyzed by both, one-way analysis of
variance and Student’s test. A p < 0.05 was considered significant.
2.5. Computational methods
2.5.1. EDMC calculations
The conformational space was explored using the method
previously employed by Liwo et al. [17] that included the EDMC
method [18] implemented in the ECEPPAK [19] package. Confor-
mational energy was evaluated using the ECEPP/3 force field [20].
Hydration energy was evaluated using a hydration-shell model with
a solvent sphere radius of 1.4 Å and atomic hydration parameters
that have been optimized using nonpeptide data (SRFOPT) [21]. In
order to explore the conformational space extensively, 10 different
runs werecarried out, each of themwith a different random number.
Therefore, a total of 5000 accepted conformations were collected.
Each EDMC run was terminated after 500 energy-minimized
conformations had been accepted. The parameters controlling the
runs were the following: a temperature of 298.15 K for the simula-
tions; a temperature jump of 50 000 K, and the maximum number of
allowed repetitions of the same minimum was 50. The maximum
number of electrostatically predicted conformations per iteration
was 400; the maximum number of random-generated conforma-
tions per iteration was 100; the fraction of random/electrostically
predicted conformations was 0.30. The maximum number of steps
at one increased temperature was 20; and the maximum number of
rejected conformations until a temperature jump was executed was
column: Lichrosorb RP C18, 7
m
m, 250 ꢁ 16 mm; gradient elution:
30e100%, 70 min; mobile phase: 80% acetonitrile, 0.1% TFA; flow
rate: 4 ml/min, 220 nm, for analytical HPLC, column: Phenomenex
Luna 5C18(2), 250 ꢁ 4.6 mm; mobile phase: 80% acetonitrile, 0.1%
TFA; flow rate: 1.2 ml/min, 220 nm, ESI-MS: Finnigan TSQ 7000.
HPLC data of the synthesized peptides
Retention
Gradient
factor (min)
elution (%)
RQIRIWFQNRRMRWRR-NH2 (2)
KQIKIWFQNKKMKWKK-NH2 (3)
RWWKWWWWWRRWKWKK-NH2 (4)
YGRKKRRQRRRC-NH2 (5)
8.808
11.510
6.026
6.898
5.775
25e40 (15 min)
20e40 (20 min)
35e50 (15 min)
12e27 (15 min)
28e43 (15 min)
HFRWRQIKIWFQNRRM[O]KWKK-NH2 (6)
The instrument for ESI-MS was Finnigan TSQ 7000 and the data
are as follows: (2) 590 (M/4þ1), 518 (M/5þ2Naþ), 495 (M/5 þ Naþ),
472 (M/5 þ1), 394 (M/6 þ 1); (3) 1081 (M/2 þ 1), 721 (M/3 þ 1), 541
(M/4 þ 1), 433 (M/5 þ 1); (4) 892 (M/3 þ 1), 670 (M/4 þ 1), 536
(M/5 þ 1); (5) 832 (M/2 þ 1), 555 (M/3 þ 1), 416 (M/4 þ 1), 356
(M/5 þ Naþ), 333 (M/5 þ 1); (6) 964.0 (M/3 þ 1), 723.2 (M/4 þ 1),
578.8 (M/5 þ 1), 482.1 (M/6 þ 1).
100. Only trans peptide bonds (
u
y 180ꢀ) were considered. All
accepted conformations were then clustered into families using the
program ANALYZE [19] by applying the minimal-tree clustering
algorithm for separation, using backbone atoms, energy threshold of
30 kcal molꢃ1, and RMSD of 0.75 Å as separation criteria. This clus-
tering step allows a substantial reduction of the number of confor-
mations and the elimination of repetitions. A more detailed
description of the procedure used here is given in section 4.4
Computational Methods of reference [13].
2.2. Microorganisms and media
Strains of C. albicans and C.neoformans from the American Type
Culture Collection (ATCC, Rockville, MD, USA) were used. C. albicans
ATCC 1 0231, and C. neoformans ATCC 32264 were grown on Sabo-
uraudechloramphenicol agar slants for 24 h at 35 ꢀC, maintained on
slopes of Sabouraudedextrose agar (SDA, Oxoid). Inocula of cell
suspensions were obtained according to reported procedures and
adjusted to 1e5 ꢁ103 cells with colony forming units (CFU)/mL [16].
2.5.2. Molecular dynamics simulations
MD simulations were performed using a combined decane/water
systemwhich was previously used with success for other compounds
of biological interest [22]. MD simulations and the trajectory analysis
were performed using the GROMACS 3.3 programs package [23]. The
GROMACS [24,25] united-atoms force field (FF) and the SPC water

372
F.M. Garibotto et al. / European Journal of Medicinal Chemistry 46 (2011) 370e377
model were used. The time step for the simulations was 0.002 ps. For
long-ranged interactions, the particle-mesh Ewald (PME) [26]
method was used with a 1 nm cutoff and a Fourier spacing of
0.12 nm. The MD protocol consisted of several preparatory steps:
energy minimization using the conjugate gradient model [27],
density stabilization (NVT conditions), and finally production of the
MDsimulation trajectory. All production simulations were performed
under NPT conditions at 300.0 K and 1.0 bar, using Berendsen’s
coupling algorithm [28] for keeping temperature (
s
T ¼ 0.1 ps) and
pressure 0.5 ps) constant. The compressibility was
(sP
¼
4.8 ꢁ 10ꢃ5 barꢃ1. All coordinates were saved every 5.0 ps. The SETTLE
[29] algorithm was used to keep water molecules rigid during MD
simulations. The LINCS[30]algorithmwasusedto constrain all length
bonds in the preparatory steps. However, no restrains were used in
production-MD simulations. A length of 50.0 ns was taken in to
account for all production-MD simulations. The molecular system
consisted of a biphasic environment in which the conformational
behaviour of peptides were examined. This biphasic system consisted
of 188 decane molecules and 1468 water molecules; the size of the
simulation box was 4.36 nm in the x and y directions, and 5.42 nm in
the z direction. This system was built up in such a way that all decane
moleculeswere forming a layerlocatednear the middleof thebox (ca.
2.71 nm in the z direction) whereas water molecules were above and
below the decane layer. The thickness of the decane layer was
w2.96 nm. Just then, the solvent (decane and water) molecules were
correctly localized into the box. An energy minimization and an NVT-
MD simulation of 10 ns long were performed in order to equilibrate
the interfaces formed between solvent molecules. After the solvent
stabilization was reached, a molecule of each peptide was embedded
into the decane/water interface. An extra step of prestabilization of
1 ns long was performed before the production-MD simulation was
started.
2.5.3. Molecular electrostatic potentials
Quantum mechanics calculations were carried out using the
Gaussian 03 program [31]. We use the most populated conforma-
tions of peptides 2ꢃ5 obtained from EDMC calculations. Subse-
quently, single point ab initio (RHF/6-31G) calculations were carried
out. The electronic study was carried out using molecular electro-
static potentials (MEP) [32]. These MEP were calculated using
RHF/6-31G wave functions and MEP graphical presentations were
created using the MOLEKEL program [33].
3. Results and discussion
On the basis of our results obtained for penetratin (RQI-
KIWFQNRRMKWKK-NH2, peptide 1) and its analogues acting as
antifungal agents [13,14], as well as on previously reported struc-
tureeactivity relationship studies performed on their penetrating
capacity [34,35], we synthesized four different analogues of pene-
tratin (compounds 2ꢃ4 and 6). Tat peptide (YGRKKRRQRRR-NH2,
peptide 5) was also included in this study. Previous reports have
indicated that the presence of R and K amino acids are important
for the antimicrobial effect of these peptides [9]. Therefore, we
synthesized and tested peptides 2 and 3 which are analogues of 1
and are rich in R and K residues, respectively. Both compounds
displayed antifungal activity against the tested fungus, particularly
against C. neoformans showing a percentage of inhibition of 77%
and 72% at a relatively low molar concentration (12.5 mM) (Table 1).
In general, peptides 2 and 3 displayed a closely related antifungal
activity with respect to that previously reported for 1.
It has been demonstrated that the presence of a significant
number of hydrophobic residues along the sequence of these
cationic antimicrobial peptides is also of importance for their bio-
logical effect. Our recent results on small-size antifungal peptides

F.M. Garibotto et al. / European Journal of Medicinal Chemistry 46 (2011) 370e377
373
[14] are an additional support for such hypotheses. Thus, we
decided to synthesize and test peptide 4, which is an analogue of
penetratin rich in Trp. However, peptide 4 displayed a lower anti-
fungal activity against the tested fungi (Table 1) compared to
penetratin.
Tat peptide (5), derived from the HIV Tat transactivator protein
[36], was identified to efficiently cross the plasma membrane. On
the basis of the above data, we decided to synthesize and evaluate
peptide 5. Our results indicated that this peptide possesses only
a marginal antifungal activity against the fungi tested here,
Fig. 1. Change in the secondary structure during molecular dynamics simulation for
peptide 6.
showing a percentage of inhibition of 100% at 100
mM against
C. neoformans (Table 1). Thus, Tat displayed a markedly lower
antifungal activity with respect to that obtained for penetratin.
We have previously reported that His-Phe-Arg-Trp-NH2, the 6-9
sequential unit of
showed antifungal properties especially against C. neoformans [37].
a
-MSH, and structurally related tetrapeptide
were obtained for the rest of peptides reported here, being the
results for peptide 1 representative of this series.
More recently we have reported that HFRWRQIKIWFQNRRMK
To corroborate these theoretical results, in the next step CD
spectroscopic measurements were performed both in water and in
a mixture of TFE and water (3:7) for peptide 6 which was the most
active compound in this series. Peptide 6 was measured at room
temperature by using the following conditions (pH adjusted by HCl/
NaOH solutions); concentration: 0.023 mM; pH ¼ 6.7.
WKK-NH2 (the 6-13 sequence of
a-MSH attached to penetratin)
displayed significant antifungal activity against both C. albicans and
C. neoformans [38]. In order to investigate if the replacement of
methionine (M) residue by methionine sulfoxide (M[O]) might
increase or decrease the antifungal effect, peptide 6 was synthesized
and tested. Peptide 6 displayed the strongest antifungal effect
against C. neoformans and a significant activity against C. albicans as
well. Thus, it should be noted that peptide 6 was the most active
peptide of this series.
In order to better understand the above experimental results,
we performed an exhaustive conformational and electronic struc-
ture study of peptides 2e6. In a first step the conformational
analysis of these peptides was carried out using EDMC calculations
[17]. These calculations were conducted in the same way as
previously reported [13,14]. EDMC results predicted that peptides
2e5 possessed a tendency to adopt helix-like secondary structures,
The spectral analysis revealed that peptide 6 showed little or no
structural preference in water (Fig. 3, black line). The “U”-type CD
spectrum reflected the presence of a very large number of different
local conformations in a time average manner. Therefore, in water,
from the shape of this U-type CD curve little characteristic
secondary structure content could be extracted for this peptide
(black line). When peptide 6 was recorded in the solvent mixture of
30% TFE and 70% H2O, a significant change in the shape of the
curves was observed. The CD curve of peptide 6 (see red line in
Fig. 3) had a spectral feature similar to those of a C-type CD curve.
Thus, the “red curve” most probably reflected a conformational
being an
a
-helix structure the most representative form for these
ensemble composed of
a- or 310-helix combined with type I/III
peptides. These conformations were characterized by hydrogen
bonds between the carbonylic oxygen (residue i) and the NH group
(residue i þ 4). The details of the EDMC results obtained for
peptides 1e5 are given in Tables S1eS5 (Supplementary data). It is
interesting to note that previous NMR studies suggested that the
Tat peptide (5) is unstructured in solution [39] which is in
disagreement with our EDMC results. In a previous work [13], we
carried out an extensive conformational study of penetratin (1)
using three different complementary approaches: molecular
mechanics, simulated annealing and MD simulations. Comparing
those results, we concluded that the three methods predicted
a helix-like structure as the preferred form for peptide 1. However,
alternative studies have suggested that this peptide in solution is
b-turns plus some percentage of still unstructured (or highly
mobile) backbone foldamers. These results demonstrated that in
the presence of a considerable amount of TFE, peptide 6 adopted an
increased amount of helical and/or type I/III
b-turn secondary
structure. These results are in agreement with those previously
reported for penetratin [34] and analogues [38].
To better characterize the spatial orientations of this peptide, we
plotted Edmundson wheel representations of peptides 1ꢃ5 (Fig. 4).
The wheel representations obtained for peptides 2 and 3 were very
similar, showing two clearly differentiated façades: the ‘charged
one’ (denoted in dash line in Fig. 4) and a more extended ‘uncharged
one’ (denoted in full line). The first face identifies the cationic resi-
dues 11, 4, 15 and 1 (R or K) as those accounting for the mutual
coulombic binding. The first three residues were located on the same
side of the helical peptide, and we named that side ‘charged face’.
These positivelycharged residuesat neutral pH wereable toproduce
salt bridges with the anionic site of lipids. The uncharged face was
more extensive and was formed by a total of six hydrophobic (M12,
I5, W6, I3, W14 and F7) and two polar residues (N9 and Q2). The rest
of the positively charged residues were homogeneously distributed.
Thus, cationic residues 16, 13 and 10 were strategically intercalated
along the ‘uncharged façade’. In relation tothe nomination used here
for the two faces, two aspects deserve an explanation. The first one is
that although the ‘uncharged face’ possesses some cationic residues,
we decided to call it ‘uncharged’ in order to differentiate this moiety,
possessing more hydrophobic and polar amino acid residues, from
the so-called ‘charged face’. The second point is that W is assumed to
be a hydrophobic aminoacid. This is not totallycorrectas W can form
hydrogen bonds and induce dipole interactions. However, for
our purposes such assumption might be considered a reasonable
approach.
either partially a-helical [40,41] or has a partial b-hairpin structure
[42]. On the other hand experimental CD studies have demon-
strated that in a membrane-mimetic environment, penetratin [34]
and analogues [38] displayed a clear tendency to form helix-like
secondary structures. In the presence of such a situation a reason-
able step to take is to look for theoretical calculations which
simulate a membrane-mimetic environment. Thus, we performed
MD simulations on a combined decane/water system which has
been previously used to simulate a membrane-like phase [22].
Fig. 1 shows the change in the secondary structure during MD
simulation for peptide 1. In this simulation the residues 5 to 12 have
shown the highest preference for a-helix conformation. In contrast,
the initial and final residues appear to have a random coil structure
because of the flexibility of these residues. Such a conformational
behaviour was observed until the end of the simulation.
Fig. 2 shows a snapshot of the peptide 6/decane/water system at
the end of the MD simulation (25 ns) displaying the spatial orien-
tation of this peptide in the membrane-like phase. Similar results

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F.M. Garibotto et al. / European Journal of Medicinal Chemistry 46 (2011) 370e377
Fig. 2. Snapshot of the location of peptide 6 taken at the end of the simulation.
The wheel representation obtained for peptide 4 was very
surface for the peptides reported here. To better appreciate the
electronic behaviour of peptide 2, and by considering that two
different faces were signalled (Fig. 4), we present the MEP of this
peptide showing both faces. Fig. 5 (top moiety) shows the ‘charged
face’, characterizedbythepresenceof the fourR residues (R1, R4, R11
and R15). Although it is possible to visualize residue R16 near this
face, this residue is somewhat shifted in direction to the uncharged
face. It has been previously reported that peptideelipid association
occurs through the formation of salt bridges between the positively
charged residues K4, R11 and K15 and the lipid phosphate groups
[43]. In addition, previous studies on tryptophan fluorescence have
similar to those of 2 and 3 with the only difference that in the case
of peptide 4 all the hydrophobic residues were W, being the
general distribution of cationic and hydrophobic residues the
same. It is interesting to note that the wheel representations
obtained for peptides 2ꢃ4 were closely related to that previously
reported for penetratin [13]. Although the antifungal activity
against C. albicans obtained for peptides 2ꢃ4 was similar to that
previously reported for penetratin, the antifungal effect obtained
against C. neoformans for peptide 4 was slightly lower with respect
to peptides 1ꢃ3. In contrast, the wheel representation obtained for
peptide 5 displayed a clearly more extended ‘charged face’,
making the general distribution of this peptide more ‘cationic’ in
relation to peptides 2ꢃ4. This is in agreement with Tat which
comprises eight cationic out of its twelve amino acid residues,
whereas penetratin and peptides 2ꢃ4 possess only seven cationic
out of the sixteen amino acids in total.
It is clear that not only the conformational aspects but the elec-
tronic properties of these peptides are determinant for the anti-
fungal activities. Thus, knowledge of the stereoelectronic attributes
and properties of these analogues of penetratin and Tat will
contribute significantly to the elucidation of the structural require-
ments to produce the antifungal activity. MEP maps are of particular
value because they allow the visualization and assessment of the
capacity of a molecule to interact electrostatically with a putative-
binding site [32]. MEP can be interpreted in terms of a stereo-
electronic pharmacophore condensing all available information on
the electrostatic forces underlying affinity and specificity. More
positive potentials reflect nucleus predominance while less positive
values represent rearrangements of electronic charges and lone
pairs of electrons. The fundamental application of this study is the
analysis of non-covalent interactions [32b], mainly by investigating
the electronic distribution in the molecule. Thus, this methodology
was used to evaluate the electronic distribution around molecular
30
25
20
15
10
5
0
-5
-10
-15
190
200
210
220
230
240
250
260
270
λ/nm
Fig. 3. The CD spectrum of peptide 6 (HFRWRQIKIWFQNRRM[O]KWKK-NH2) in water
(black line) and in TFE/H2O (3:7) (red curve) (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article).

F.M. Garibotto et al. / European Journal of Medicinal Chemistry 46 (2011) 370e377
375
Fig. 4. Edmundson wheel representations of peptides 1ꢃ5. The number in the center of the wheel corresponds to the peptide number. The “charged” and “non-charged” faces are
shown in blue dash lines and full green lines, respectively. Positively charged amino acids are denoted with blue dots, the polar with orange and the hydrophobic with yellow (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
shown the importance of peptide positivelycharged residues for the
initial binding to negatively charged vesicles since double R/K/A
mutations, involving residues K4/R10/R11/K13/K15 significantly
decreased the binding affinity [44]. The MEP of 2 suggests that the
above mentioned residues (R1, R4, R11 and R15) might be respon-
sible for the initial binding. Although the main positive potentials (V
(r) ranging from 0.73 to 0.47 el.auꢃ3) are concentrated on this side, it
should be noted that there is a relatively homogeneous distribution
of positively charged residues along the entire helical structure.
Thus, residues R16, R13 and R10 are strategically located in an
alternated manner within the uncharged face. Fig. 5 (bottom
moiety) displays the ‘uncharged face’. It appears that a kind of
cation-
The cation-
tions is recognized as an important non-covalent binding interac-
tionwhich may commonly occur within -helices [45]. Lensink et al.
p
interaction through W6/R10 takes place in this portion of 2.
p
interaction between residues at the i and (i þ 4) posi-
a
[43] reported that hydrophobic residues could protect the peptide
from the water phase. A clear hydrophobic interaction between I3
and W6 might also be appreciated in Fig. 5 (bottom moiety). It
appears that charge neutralization is required for a deeper insertion
of these peptides into the hydrophobic core of the membrane. The
extended uncharged face alternating cationic residues among the
hydrophobic one observed in the MEP of these peptides appears to
be operative in this sense.
Fig. S1 and S2 (in Supplementary data) show the MEP obtained
for peptides 3 and 4, respectively. The MEP of penetratin (1) was
also included in this analysis for comparative purposes (Fig. S3 in
Supplementary data). This MEP was previously reported in refer-
ence 13; however, in this work this MEP was recalculated and
plotted using the same scale used for the rest of peptides reported
here. Comparing the MEP obtained for peptides 1ꢃ4, it is clear that
all of them possess a closely related electronic distribution which
might explain, at least in part, the similar antifungal activity
obtained for these peptides.
Fig. 5. Electrostatic potential-encoded electron density surfaces of peptide 2: ‘charged
face’ (top moiety) and ‘non-charged face’ (bottom moiety). The surfaces were gener-
ated with GAUSSIAN 03 using RHF/6-31G single point calculations. The coloring
electrostatic potential in red is indicating the strongest attraction to a positive point
charge whereas blue is indicating the strongest repulsion. The electrostatic potential is
the energy of interaction of the positive point charge with the nuclei and electrons of
a molecule. It provides a representative measure of overall molecular charge distri-
bution. The colour-code is shown at the left (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 6 shows the MEP obtained for peptide 5. This surface
displays a more extended positively charged face with respect to

376
F.M. Garibotto et al. / European Journal of Medicinal Chemistry 46 (2011) 370e377
[46]. However, we have not obtained definitive results about the
possible molecular mechanism for these peptides yet.
4. Conclusions
In the present paper, we report the synthesis and antifungal
effects of penetratin analogues and Tat peptide. In addition,
a conformational and electronic study of these peptides was carried
out. Our conformational study from experimental (CD spectros-
copy) and theoretical methods (MD calculations using a combined
decane/water system simulating the biological environment)
indicated that these peptides adopt a helical secondary structure as
the preferred form in an environment of low polarity.
Theanaloguesofpenetratinreportedhere(compounds2ꢃ4and6)
displayed antifungal effect against C. albicans and C. neoformans being
peptide 6 (HFRWRQIKIWFQNRRM[O]KWKK-NH2) the most active
peptide in this series. In contrast, Tat (peptide 5), a well-known cell
penetrating peptide, do not show a significant antifungal activity
against fungus tested here. It appears that the lack of hydrophobic
residues in this peptide might be responsible for its lower bioactivity.
The antifungal activity obtained for these peptides is very
interesting per se, but considering their cell penetrating properties,
they are also excellent candidates to be used as carriers of other
antifungal compounds, thus enhancing their pharmacokinetic
aspects.
In addition, the results reported here provide an additional
support for the pharmacophoric patron previously reported for
penetratin and its derivatives. This pattern suggests a particular
combination of cationic and hydrophobic residues, adopting
a definite spatial ordering which appears to be the key factor for the
transition from the hydrophilic to the hydrophobic phase. This
transition appears to be a necessary step to produce the antifungal
activity. We believe that our results might contribute to the
understanding of the minimal structural requirements for the
antifungal effects of peptides reported here.
Fig. 6. Electrostatic potential-encoded electron density surfaces of peptide 5: ‘charged
face’ (top moiety) and ‘non-charged face’ (bottom moiety).
the other peptides. Eight cationic residues, namely R3, K4, K5, R6,
R7, R9, R10 and R11 are located in this section. All of them, except
K5 might be observed in Fig. 6 (top moiety). A very extensive deep
blue zone with potential values in the order of 0.70 el auꢃ3 clearly
dominates this surface. Fig. 6 shows that peptide 5 is ‘too cationic’
with respect to penetratin and peptides 2ꢃ4. Previously reported
MD simulations indicated that the aromatic residues did not
contribute to the initial binding, but rather to the subsequent
insertion of penetratin between the bilayer head groups, when they
shielded the peptide from the aqueous phase [43]. The importance
of hydrophobic residues seems to be crucial for the antifungal
activity as well. Thus, the different electronic distribution displayed
for peptide 5 with respect to the other peptides might be respon-
sible for its lower antifungal activity. These results are in agreement
with those previously reported for other so-called ‘too cationic’
peptides, displaying only marginal antifungal activity [13]. These
results are an additional support for the key role of the hydrophobic
residues in these peptides acting as antifungal compounds.
Acknowledgements
This work is part of the HungarianeArgentine Intergovern-
mental S&T Cooperation Programme. This research was partially
supported by grants from Universidad Nacional de San Luis and it is
part of the Iberoamerican Project X.7 PIBEAFUN (Search and
development of new antifungal part of the Iberoamerican Program
of Science and Technology for the Development (CYTED)). R.D.E. is
a member of the CONICET (Argentina) staff.
Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2010.10.025.
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Nevertheless, they allowed the identification of a promising 3D
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mechanism of action of the peptides reported here, in general
cationic peptides possess a relatively non-specific mechanism of
action, either causing a detergent-like disruption of the bacterial or
fungal cell membrane or forming transient transmembrane pores
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