Structure-antifungal activity relationship of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 and analogues

Article abstract of DOI:10.1016/j.bmc.2008.02.072

The synthesis, in vitro evaluation and conformational study of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ and analogues acting as antifungal agents are reported. Among them, His-Phe-Lys-Trp-Gly-Arg-Phe-Val-NH₂ exhibited a moderate but significant antifungal activity against Cryptococcus neoformans, Candida albicans and Candida tropicalis. A theoretical study allows us to propose a biologically relevant conformation for these octapeptides acting as antifungal agents. In addition, these theoretical calculations allow us to determine the minimal structural requirements to produce the antifungal response and can provide a guide for the design of compounds with this biological activity.

Full text of DOI:10.1016/j.bmc.2008.02.072

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4347–4358
Structure–antifungal activity relationship
of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 and analogues
a
*
Marcelo F. Masman,a,b Csaba Somlai,c Francisco M. Garibotto,a,b Ana M. Rodrıguez,
´
Agustina de la Iglesia,d,e Susana A. Zacchino,e Botond Penkec and Ricardo D. Enriza,b,
aDepartamento de Quı´mica, Facultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis,
Chacabuco 915, 5700 San Luis, Argentina
bIMIBIO-CONICET, UNSL, Chacabuco 915, 5700 San Luis, Argentina
cDepartment of Medical Chemistry, University of Szeged, Do´m ter 8, H-6720 Szeged, Hungary
dMicrobiologı´a, Facultad de Medicina, Universidad Nacional de Rosario, Santa Fe 3100, 2000 Rosario, Argentina
eFacultad de Ciencias Bioquı´micas y Farmace´uticas, Farmacognosia, Universidad Nacional de Rosario,
Suipacha 531, 2000 Rosario, Argentina
Received 6 November 2007; revised 20 February 2008; accepted 22 February 2008
Available online 4 March 2008
Abstract—The synthesis, in vitro evaluation and conformational study of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 and analogues
acting as antifungal agents are reported. Among them, His-Phe-Lys-Trp-Gly-Arg-Phe-Val-NH2 exhibited a moderate but significant
antifungal activity against Cryptococcus neoformans, Candida albicans and Candida tropicalis. A theoretical study allows us to pro-
pose a biologically relevant conformation for these octapeptides acting as antifungal agents. In addition, these theoretical calcula-
tions allow us to determine the minimal structural requirements to produce the antifungal response and can provide a guide for the
design of compounds with this biological activity.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the broad use of antifungal agents.5 As a consequence,
there is an urgent need of a next generation of new anti-
fungal agents, which may overcome the above
disadvantages.
The prevalence of systemic and dermal fungal infections
has significantly increased during the past two decades
and remains an important cause of great morbi-mortal-
ity of immunocompromised patients.1 In a recent study,
McNeil et al.2 found a dramatic increase in mortality
from 1980 to 1997 due to mycoses originated in multiple
causes which were mainly associated with Candida,
Aspergillus and Cryptococcus spp. infections. Added to
these fungal spp., new emerging fungal pathogens ap-
pear every year as the cause of morbidity and life-threat-
ening infections in the immunocompromised hosts.3,4
Natural and synthetic peptides have gained attention as
potential new antifungal agents.6–9 They have shown to
inhibit a broad spectrum of pathogens and microorgan-
isms10–12 and possess the important characteristic that
they do not usually induce bacterial or fungal resistance.13
Most of these peptides are believed to exert their antimi-
crobial activities either forming multimeric pores in the li-
pid bilayer of the cell membranes,13 or interacting with
DNA or RNA after penetration into the cell.14–16 In con-
trast, a-melanocyte stimulating hormone (a-MSH) and
its C-terminal tripeptide Lys-Pro-Val, which showed anti-
microbial activity against the two representative patho-
gens Staphylococcus aureus and Candida albicans,17
appear to act through a mechanism substantially different
from that of other natural antimicrobial peptides.
Although different antifungal agents are available for
the treatment of fungal infections, some of them have
undesirable side effects, are ineffective against new or
re-emerging fungi or develop resistance mainly due to
Keywords: a-MSH analogues; Octapeptides; Antifungal peptides;
Conformation; Electronic properties.
Catania et al. have reported the antifungal activity of
several peptides.17–20 They have indicated that the
antimicrobial effects of a-MSH are exerted through a
*
Corresponding author. Tel.: +54 2652 423789; fax: +54 2652
431301; e-mail: denriz@unsl.edu.ar
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.072

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M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
unique mechanism different from that of other natural
peptides, suggesting that the candidacidal effect of
these compounds is linked to the cAMP-inducing activ-
ity. As part of our ongoing program aimed at identify-
ing novel antifungal compounds,21–23 we have recently
reported the synthesis and antifungal properties of
His-Phe-Arg-Trp-NH2 and structurally related tetra-
peptides.24 In this paper, we report the synthesis and
conformational analysis of His6-Phe7-Arg8-Trp9-Gly10-
Lys11-Pro12-Val13-NH2 (1), which is the 6–13 sequence
of a-MSH and structurally related octapeptides with
antifungal properties. The aim of our work is to devel-
op more potent antifungal peptides and to understand
the conformational features that enhance potency and
selectivity and their relation to the amino acid se-
quence. For this purpose, ten new analogues with sin-
gle or double replacements within the a-MSH(6–13)
sequence were designed, prepared and tested (peptides
2–11 in Table 1).
In addition, an exhaustive conformational analysis and
an electronic properties study on all the peptides re-
ported here was carried out in order to determine a
possible biologically relevant conformation for these
compounds acting as antifungal agents.
2. Results and discussion
The principal goal was to find a-MSH analogues with
greater antifungal activity and to reach a better under-
standing of the peptide structure–antifungal relationship
through design, synthesis and testing of novel octapep-
tide analogues in which several modifications were
made. As starting structure, we chose the sequence
a-MSH(6–13), His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2,
which contains the invariant ‘core’ sequence His-Phe-
Arg-Trp (6–9), common to all melanocortin receptors,25
and the sequence Lys-Pro-Val (11–13), which has been
reported to be relevant to antimicrobial activity.17 Previ-
ously, we tested the antifungal activity of tetrapeptide
His-Phe-Arg-Trp-NH2 and Lys-Pro-Val-NH2 against a
panel of pathogenic fungi.24 Lys-Pro-Val-NH2 did not
show antifungal activity; in contrast, the tetrapeptide
His-Phe-Arg-Trp displayed a moderated but significant
antifungal effect, in particular against C. neoformans.24
To better characterize the structure–antifungal activity
relationship of octapeptides 1–11 reported here, the
present research explored influences of amino acid sub-
stitutions on its antifungal activity.
For the a-MSH activity, the importance of the pres-
ence of His-6 has been previously demonstrated by
the fact that Ac-a-MSH7–10-NH2 lacked measurable
activity.25 Trp-9 was also critical for activity as demon-
strated by the lack of biological activity of Ac-a-
MSH6–8-NH2.25 More recent structure–activity rela-
tionship (SAR) studies including physiological effects
of a-MSH might be obtained from references.26–29 Re-
cently, we reported that the presence of His-6 and Trp-
9 residues appears to be a structural requirement for
the antifungal activity as well. In contrast, our results
indicated that Tyr and Lys could replace Phe-7 and
Arg-8, respectively.24 It is well known that the receptor
binding and transducing properties of melanocortins
depend on separate structural and conformational
characteristics.30 For example, Phe-7 plays a key role
in receptor binding whereas Lys-11 and Pro-12 are
more important for receptor stimulation.30 Thus, the
positions 7, 8, 11 and 12 were systematically mutated
in the series presented here. The resulting peptides were
tested against three human pathogenic strains (C. albi-
cans, Cryptococcus neoformans and Candida tropicalis).
2.1. Antifungal activity
According to Section 4, concentrations of peptides at
100 and 200 lM were incorporated to growth media in
order to carry out the antifungal evaluation. Com-
pounds producing no inhibition of fungal growth at
200 lM level were considered inactive.
In a first step of our study, we focused our attention on
the C-terminal moiety (a-MSH(11–13) sequence), syn-
thesizing and evaluating compounds 1–4. Our results
Table 1. Antifungal activity (% inhibition = mean SD) of peptides against Candida albicans ATCC 10231, Candida tropicalis C 131 2000 and
Cryptococcus neoformans ATCC 32264
Peptide
Sequence
Cryptococcus
neoformans
Candida albicans
Candida tropicalis
% inhib.
200 lM
% inhib.
100 lM
% inhib.
200 lM
% inhib.
100 lM
% inhib.
200 lM
% inhib.
100 lM
1
His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
His-Phe-Arg-Trp-Gly-Lys-Phe-Val-NH2
His-Phe-Arg-Trp-Gly-Lys-Tyr-Val-NH2
His-Phe-Arg-Trp-Gly-Arg-Phe-Val-NH2
His-Phe-Lys-Trp-Gly-Lys-Phe-Val-NH2
His-Phe-Lys-Trp-Gly-Arg-Phe-Val-NH2
His-Phe-Lys-Trp-Gly-Lys-Tyr-Val-NH2
His-Tyr-Lys-Trp-Gly-Arg-Phe-Val-NH2
His-Tyr-Lys-Trp-Gly-Lys-Tyr-Val-NH2
His-Tyr-Lys-Trp-Gly-Lys-Phe-Val-NH2
His-Tyr-Arg-Trp-Gly-Lys-Phe-Val-NH2
24 5
77 30
9
2
0
0
0
0
2
15
58
72
38
98
50
47
42
25
21
100
3
1
7
6
3
6
4
1
1
7
61 23
43 10
80 0.6
5
2
19 10
21 10
64 12
9
3
3
3
99
94
97
97
1
6
3
4
0
0
4
1
0.2
1
1
5
72
100 0.4
8
0
22
92
0
5
0
6
80
0
4
58
0
6
7
83 20
89
15 12
8
5
11
0
3
0
23
3
8
0
9
10
63 15
41 10
35 10
100
0
3
2
3
0
0
0
1
0
11
Amph B
10
100
6
0
100
1
100
0
100
The main mutation performed in the peptide sequences and the percentage of inhibition higher than 50, are denoted in bold.
Amph B, amphotericin B.

M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
4349
showed that octapeptide 1 did not inhibit C. albicans up
to a concentration = 200 lM (Table 1). This result is in
contrast to that of Grieco et al.19 who reported that
peptide 1 possessed a moderate inhibitory activity
against C. albicans (59.4%) at 100 lM. Peptide 1 did
not inhibit C. tropicalis either, but showed a low inhib-
itory effect against C. neoformans (24%) at 200 lM. The
replacement of Pro-12 by Phe in peptide 2 produced an
enhancement of the antifungal activity against all fungi
tested mainly at 200 lM. These results were in agree-
ment with those of Grieco et al.19 who reported a high-
er inhibitory effect for peptide 2 in comparison to
peptide 1 against C. albicans. The replacement of Pro-
12 of peptide 1 by Tyr in peptide 3 gave a higher
enhancement of activity (compare 3 with 2 and 1) only
against C. neoformans. We then replaced Lys-11 in 2 by
Arg obtaining peptide 4, which displayed an enhance-
ment of the antifungal activity at 200 lM in the three
fungi tested.
In order to have an overview of the potential of these
peptides as antifungal agents, we show the percentage
of inhibition of all compounds at concentrations
below 100 lM, in comparison to the behaviour of
amphotericin B. Peptides 1–11 were almost devoid of
activity against species of Candida genus between 50
and 0.4 lM (results not shown), but they possess mod-
erate percentage of inhibition against C. neoformans
(Table 2).
In order to better understand the above experimental
results, we performed an exhaustive conformational
and electronic properties study of all the peptides re-
ported here using theoretical calculations. These re-
sults are presented in the next section and might be
useful to perform a structure–activity relationship on
this series.
2.2. Theoretical calculations
In a second step of our study, we focused our efforts
on the introduction of modifications in the sequence
a-MSH(6–9). Replacement of Arg-8 by Lys in 2 and
4 gave peptides 5 and 6, respectively. This change pro-
duced a significant enhancement (p < 0.01) of anti-
fungal activity, from 15% (peptide 2) to 38%
(peptide 5) and from 72% (peptide 4) to 98% (pep-
tide6) of inhibition at 100 lM against C. neoformans.
The same trend was observed for compounds 5 and 6
when tested against C. albicans and C. tropicalis. It is
interesting to emphasize that peptide 6 was the most
active peptide in this series, showing a significant anti-
fungal activity against all the fungi tested, including
C. albicans. Next, we replace Phe by Tyr-12 in peptide
5 obtaining peptide 7. Although peptide 7 displayed a
similar activity against C. neoformans to that of 5 and
6 at 200 lM, its inhibitory effect against this yeast at
100 lM, as well as its activity against C. albicans
and C. tropicalis was markedly lower. Peptides 8–11
were obtained replacing Phe-7 by Tyr in peptides 6,
7, 5 and 2, respectively. In general, these peptides dis-
played a lower inhibitory effect with respect to their
congeners, in particular peptides 10 and 11, which dis-
played only a marginal effect.
2.2.1. Conformational study. Linear peptides are highly
flexible and, therefore, to determine the biologically rel-
evant conformations is not an easy task. It is necessary
to perform an exhaustive conformational analysis for
these structures to obtain a good perspective about their
conformational intricacies. Thus, a conformational
study on all peptides reported here was carried out using
EDMC calculations.31,32 Theoretical calculations were
carried out as described in calculation methods section.
These results are summarized in Table 3 and Tables S1–
S11 (as Supplementary material).
Calculations yielded a large set of conformational fam-
ilies for each peptide studied. The total number of con-
formations generated for each peptide varied between
94,082 and 110,569, and the number of those accepted
was 5000 for all cases. In the clustering procedure, a
˚
route mean square deviation (RMSD) of 0.75 A and a
DE of 30 kcal molꢀ1 were used. The number of families
after clustering varied between 221 and 366. The total
number of families accepted with a relative population
higher of 0.20% varied between 8 and 20. Their popula-
tions summed up to ca. 90% of all conformations in each
case (see Table 3).
Table 2. % of inhibition of Cryptococcus neoformans by peptides 1–11 at concentrations between 50 and 0.4 lM
Peptide
% inhibition
6.25 lM 3.25 lM
50 lM
25 lM
12.5 lM
1.62 lM
0.81 lM
0.40 lM
1
0
15
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
41 0.6
1
0
24
0
3
56 18
67 19
28
26
0
5
3
9
10
12
0
5
4
6
4
4
26
0
5
18 10
0
11
0
3
10
0
2
7
1
5
23
96
2
2
1
0
36
6
95 0.7
49 0.4
n.t.
84
1
84
31
19
10
0
3
3
8
2
57 8
36 10
45
33
21
n.t.
0
4
1
3
32
31
2
8
5
7
7
50
n.t.a
40 0.1
n.t.
26 0,2
0
8
17
n.t.
0
4
2
9
10
39 17
22
38
3
2
1
19
34
2
1
6
n.t.
0
n.t.
0
43
37
2
6
11
Amph Bb
24
100
21
100
16
100
7
16
100
7
5
100
3
0
100
0
100
100
a n.t., not tested.
b Amphotericin B.

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M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
Table 3. Selected conformational search and clustering results for peptides 1–11 optimized at the EDMC/SRFOPT/ECCEP/3 level of theory
Generateda
Acceptedb
# Fc
# F0.20%
% Pe
d
Peptide
Electrost.
Random
Thermal
Total
Electrost.
Random
Thermal
Total
1
7051
6450
7165
7277
6372
6525
7437
7419
7032
7547
6752
95,871
88,021
96,921
96,253
87,496
87,475
10,1014
100,395
96,579
102,709
92,559
237
181
258
275
214
208
267
317
258
313
227
103,159
94,652
650
501
552
622
565
468
628
500
553
530
571
4158
4356
4246
4169
4266
4378
4164
4261
4284
4218
4247
192
143
202
209
169
154
208
239
163
252
182
5000
5000
5000
5000
5000
5000
5000
5000
5000
5000
5000
271
254
221
298
252
272
274
305
227
366
283
15
20
8
92.16
92.86
92.48
91.38
93.06
92.08
92.00
91.52
93.84
88.74
92.60
2
3
104,344
103,805
94,082
4
16
13
14
16
13
12
20
16
5
6
94,208
7
108,718
108,131
103,869
110,569
99,538
8
9
10
11
a Number of conformations generated electrostatically, randomly and thermally during the conformational search.
b Number of conformations accepted from those generated electrostatically, randomly and thermally during the conformational search.
c # F, total number of conformational families a as result of the clustering run.
d # F0.20%, number of conformational families with populations above 0.20%.
e % P, sum of the percent relative population of # F0.20%
.
Our results indicate that these peptides are highly flexi-
ble, showing equilibrium of statistical-coil33 structures.
All low-energy conformers of octapeptides studied here
were then compared to each other. The comparison in-
volved the spatial arrangements, relative energy and
populations.
populations obtained for the fully-folded and partially
extended forms were in equilibrium, giving at least
50% of the whole population. It is interesting to note
that peptide 5, which was one of the most active peptides
in this series, displayed the partially extended conforma-
tion as the highly preferred form with 65% of the whole
population. In contrast, peptides 1, 10 and 11, which
were inactive or displayed only marginal activity, did
not adopt the partially extended conformation among
the preferred forms. These peptides displayed the fully-
folded form as the highly preferred conformation, and
a mixture between bend and coil conformations, as the
second preferred form (see % of population in Tables
S1, S10 and S11 in Supplementary material). Thus,
our results permit us speculate that the low antifungal
activity obtained for peptides 1, 10 and 11 could be ex-
plained at least in part by this conformational difference.
Interestingly enough, in the fully-folded conformations,
a stabilizing p-stacking interaction between the aromatic
residue at 7 position (Phe-7 or Tyr-7) and the other aro-
matic residue located at 12 position (Phe-12 or Tyr-12)
was observed.
The preferred fully-folded form with 42% of population
(global minimum) obtained for peptide 1 is comparable
to the conformation reported by Caratenuto et al.20
from RMN spectroscopic data. The torsional angles ob-
tained for the fragment Arg8-Trp9-Gly10-Lys11-Pro12 are
the following: /8 = ꢀ65 (ꢀ67 5), w8 = ꢀ41 (ꢀ41 3);
/9 = ꢀ89 (ꢀ76 3), w9 = ꢀ29 (ꢀ26 2);
/10 = 94
(ꢀ84 5), w10 = 39 (ꢀ1 15); /11 = ꢀ63 (ꢀ67 13),
w
11 = ꢀ58 (ꢀ46 6); /12 = ꢀ68, w12 = ꢀ19 (ꢀ3 45).
The experimental data are given in brackets. As can be
observed, there is a very good correlation between the
theoretical and experimental dihedral angles for Arg-8,
Trp-9, Lys-11 and Pro-12. Nevertheless, the values ob-
tained for Gly-10 are quite shifted. Whereas from the
theoretical calculations it appears that this residue
adopts a bend structure, the experimental results suggest
a turn form. However, it must be pointed out that Gly is
a very flexible residue as was previously highlighted by
Carotenuto et al.20 and therefore this difference is not
unexpected.
Molecular recognition and the converse concept of spec-
ificity34 are explained in mechanistic and reductionistic
terms by a stereoelectronic ‘complementarity’ between
the ligand and the receptor.35 In this context, it is obvi-
ous that the knowledge of the stereoelectronic attributes
and properties of octapeptides and its analogues will
contribute significantly to the elucidation of a possible
pharmacophoric patron for these compounds acting as
antifungal compounds. Once the low-energy conforma-
tions for the octapeptides reported here were obtained
and, in an attempt to find the potentially reactive sites
for the ligands, we evaluated the electronic aspects of
the molecules under study using molecular electrostatic
potentials (MEP).36
It was found that the active octapeptides 2–9 possess
low-energy conformers which were similar to each other.
They showed mainly two preferred conformations con-
sidering the relative energy and population. These were
a fully-folded conformation and a partially extended
form (see Fig. 1). The fully-folded conformation dis-
played a turn along 7–10 residues, a bend at residue
11 and the rest of the residues (6, 12 and 13) in a non-
stable structure (Fig. 1a). The partially extended confor-
mation showed an extended form encompassing the first
three residues, a turn along 9–12 residues and the last
residue in a non-stable structure (Fig. 1b). In addition,
peptides 7–9 displayed an a-helix conformation with a
relatively relevant population. In general, the relative
2.2.2. Molecular electrostatic potentials (MEP). MEP 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.36–38 MEP

M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
4351
Figure 1. Spatial view of the preferred forms obtained for peptide 6: (a) fully-folded conformation and (b) partially extended conformation. The
backbones are shown as balls and sticks whereas the side chains are shown as wire representations. All hydrogens are omitted for clarity.
can be interpreted in terms of a stereoelectronic pharma-
cophore condensing all available information on the
electrostatic forces underlying affinity and specificity.
tides 3, 7–11 may be, in principle, related to the stronger
stabilizing p-stacking interaction present in these struc-
tures. It appears that the replacement of Phe-7 or Phe-
12 by Tyr could be the responsible for such difference.
It is interesting to note that the most active peptides
(4–6) possessing preferred partially extended forms, do
not have Tyr in their respective sequence.
Figure 2 gives the MEP obtained for compounds 1 and 3,
showing the C-terminal portion of these peptides. The
different electronic behavior obtained for this moiety of
peptide 1 with respect to 3 and the rest of the octapep-
tides reported here can be appreciated in this figure. Note
that the electrostatic potentials near to the C-terminal
portion of 1 are markedly more negative than those ob-
tained for peptide 3. Thus, there is not only a different
conformational behavior between peptide 1 and the rest
of peptides but also a clear different electronic distribu-
tion due to the replacement of an aromatic residue
(Phe or Tyr) by Pro-12. The different conformational
and electronic behavior observed for 1 could explain
the lack of antifungal effect observed for this peptide.
2.2.3. Structure–antifungal activity relationships. In a
previous paper we demonstrated that the smallest frag-
ment with measurable antifungal activity was His6-
Phe7-Arg8-Trp9-NH2 and some of its derivatives.24 It
has been shown that the minimal sequence possessing
a-MSH-like activity is the central tetrapeptide His-
Phe-Arg-Trp.25 This ‘core’ sequence was suggested to
be the ‘message’ fragment for a-MSH, the rest of the
molecule being regarded as the ‘address’ sequence.39
Keeping in mind that peptides 2–9 can be considered ac-
tive, whereas peptides 1, 10 and 11 have reduced inhib-
itory effect, it is noteworthy that the partially extended
conformations as preferred forms were only present in
active analogues, whereas fully-folded conformations
were present both in active and inactive peptides. This
suggests that the partially extended conformations could
be operative in the recognition process between these
peptides and their receptor. Figure 4 shows a spatial
view of the most populated and energetically preferred
conformation of peptide 5. This conformation has been
overlapped with the partially extended conformations of
the rest of the active peptides 2–4 and 6–9. The conform-
ers shown in Figure 4 are very close to each other con-
sidering the values of the dihedral angles obtained for
their backbones of the N-terminal portion (residues 6–
9). From the analysis of Figure 4, it is clear that there
Figure 3 gives the MEP obtained for peptides 5, 9 and
10 showing, in this case, a spatial view of the stabilizing
p-stacking interaction between the aromatic residues lo-
cated at positions 7 and 12. The p-stacking interaction
between Phe-7 and Phe-12 in peptide 5 (Fig. 3a) displays
both aromatic residues in a parallel orientation and it is
located far away in comparison to the similar interac-
tion observed in peptides 9 and 10 (Fig. 3b and c, respec-
tively). It should be noted that the p-stacking
interactions involving Tyr residues (either at positions
7 or 12) adopted a perpendicular orientation between
the aromatic residues and also displayed a nearby spa-
tial position. These results suggest that p-stacking inter-
actions involving Tyr residues appear to be stronger
than those in which only Phe residues participate. The
preference for fully-folded structures observed for pep-

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M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
Figure 2. Electrostatic potential-encoded electron density surfaces of peptides 1 (a) and 3 (b). The surfaces were generated with Gaussian 03 using
RB3LYP/6-31G(d,p) single point calculations. The coloring represents electrostatic potential with red indicating the strongest attraction to a positive
point charge and blue 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 distribution. The color-coded is shown on the left.
is not a complete conformational overlapping between
these conformers. However, all the active peptide
adopted a very similar spatial ordering for the ‘core’
6–9 sequence. A good fit among the four first amino
acids was obtained, but there was not a good overlap-
ping between the last four amino acids (residues 10–
13). Thus, the backbone conformations of C-terminal
tripeptide for the partially extended forms do not appear
to be essential for the general spatial organization of
these octapeptides. This situation might be well appreci-
ated in Figure 4. In contrast, our results indicate that in
the fully-folded forms of these peptides, the spatial
ordering adopted by the C-terminal tripeptide (11–13 se-
quence), in particular, the aromatic amino acid Phe-12
or Tyr-12 could play a determinant role for the whole
conformation of the octapeptide making a stabilizing
p-stacking interaction.
These results are in good agreement with our previous
ones postulate a partially extended conformations as
the biologically relevant form for His-Phe-Arg-Trp-
NH2 and analogues.24 Interestingly, the acetylation of
the same peptides led to inactive compounds possessing
a highly preferred fully-folded conformation.24
Besides, these results agree fairly well with those previ-
ously reported by Sugg et al.40 postulating a partially ex-
tended conformation for the His-Phe-Arg-Trp ‘core’
sequence as the biologically active a-MSH conforma-
tion. Thus, it is worthwhile to compare the ‘biologically
active conformation’ proposed in this study for the
backbone of the His-Phe-Arg-Trp-NH2 portion, with
that previously reported for the same sequence from
proton NMR studies in aqueous solution.40 Sugg et al.
reported that the observed topology was consistent with

M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
4353
Figure 3. Electrostatic potential-encoded electron density surfaces of peptides 5 (a), 9 (b) and 10 (c). The color-coded is shown on the left.
a non-hydrogen-bonded b-like structure (/ = ꢀ139ꢁ and
w = 135ꢁ for L-amino acids) as the predominant confor-
mation in solution. This conformation is closely related
to those obtained for the ‘core portion’ (6–9 sequence)
of the different conformers of the active peptides. In Fig-
ure 5, we overlapped the conformation reported by Sugg
et al. (in orange) with the partially extended conforma-
tions obtained for all the active peptides.
contrast, our results indicate that the presence of Pro-
12 is not relevant for the antifungal activity. Replace-
ment of Pro-12 by Phe or Tyr gives a clear enhancement
of inhibitory effect. A striking difference can be observed
between the roles of Pro-12 of these peptides when act-
ing as anti-inflammatory or antifungal compounds. On
the other hand, the ‘core’ 6–9 sequence (His-Phe-Arg-
Trp) of melanocortins has long been considered essential
for melanocortin receptor recognition and activation.43
Alanine replacement studies in B16 melanoma cells
showed that when each amino acid in the 6–9 sequence
was replaced by alanine there was a substantial decrease
in receptor recognition and activation by a-MSH.44 Our
results indicate that the ‘core’ 6–9 sequence, or a very
similar structure, is also a structural requirement to pro-
duce the antifungal activity.
Grieco et al.17–20 suggested that the antimicrobial effects
of a-MSH could be exerted through a unique mecha-
nism different from that of other natural antimicrobial
peptides. They suggested that the candidacidal effect of
a-MSH is linked to the cAMP-inducing activity using
toluene permeabilized cells. They proposed that there
is some parallelism between the a-MSH and antifungal
effects. Our results, using entire cells, showed some par-
allelism with those previously reported by Grieco et al.;
however, it should be noted that there are interesting
differences as well. Previous structure–activity studies
on the a-MSH(11–13) sequence disclosed the signifi-
cance of Pro-12 to the anti-inflammatory influence of
the tripeptide.41 Indeed, substitution of Pro-12 with its
D-isomer abolished the anti-inflammatory effect.41 Sub-
sequently, researches on C-terminally modified ana-
logues of a-MSH confirmed the important role of Pro-
12 for binding and activity at the MC1 receptor.42 In
Carotenuto et al.20 hypothesized that the N-terminal
portion (7–10) is responsible for receptor activation
while the C-terminal moiety (10–13) works only enforc-
ing the binding to the receptor through positive interac-
tions with an auxiliary-binding site, for the candidacidal
activity.20 Our results are in a relatively good agreement
with such hypothesis. It is clear that the fully-folded
conformations, stabilized by p-stacking interactions,
do not show an adequate spatial ordering for the hypo-
thetical receptor possessing two-binding sites. On one

4354
M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
Figure 4. Stereoview of the overlapping of peptides 2–9 partially extended conformers. The backbone ‘core’ 6–9 sequence is shown in sticks
indicating each alpha carbon with spheres, the rest of the backbone structures are shown in wire representations. Peptide 2 is shown in red, 3 (blue), 4
(white), 5 (pink), 6 (cyan), 7 (yellow), 8 (green) and 9 (purple). All hydrogens and side chains are omitted for clarity.
hand, they cannot reach the adequate distances for both
interactions and, on the other hand, the intra-molecular
interactions could affect the binding of the N-terminal
portion proposed as responsible for the receptor activa-
tion. A similar explanation could be applied for the inac-
tive acetylated derivatives previously reported.24 The
fact that His-Phe-Arg-Trp-NH2 is the smallest fragment
with significant antifungal activity could be an addi-
tional support to consider the N-terminal portion as
responsible for receptor activation. However, caution
is required for such speculations, in particular, because
we are hypothesizing on a still unknown receptor.
Although the actual existence of melanocortin receptors
in yeasts has not been established, our results showing
the effects of amino acid substitutions on critical por-
tions, suggest that the presence of such receptors or
more probably a structurally related receptor is highly
plausible.
C-terminal sequences and tested their antifungal activ-
ity. Among compounds tested, His-Phe-Lys-Trp-Gly-
Arg-Phe-Val-NH2 (6) displayed the best antifungal
activity against C. neoformans, C. albicans and C. tropi-
calis. Other octapeptide analogues reported here dis-
played moderate but still significant antifungal effects
against C. neoformans.
A detailed conformational and electronic properties
study supported by theoretical calculations helped us
to identify a possible ‘biologically relevant conforma-
tion’ and also understand the minimal structural
requirements for the antifungal actions of octapeptides
reported here. Our results are very encouraging since
they show a great potential of His-Phe-Lys-Trp-Gly-
Lys-Phe-Val-NH2 and its derivatives as a truly novel
class of antifungal compounds particularly against the
yeast C. neoformans.
The general picture emerging from our results is that
there is some parallelism between the recognition pro-
cess for the a-MSH receptor and the hypothetical recep-
tor involved in the antifungal response. However, in this
regard it is interesting to note that there are also struc-
3. Conclusions
We designed and synthesized 11 a-MSH octapeptide
analogues with different residues in the core and in the

M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
4355
Figure 5. Stereoview of the overlapping of the backbone ‘core’ 6–9 sequence conformation from Ref. 40 in orange sticks, and the partially extended
backbone conformers of peptides 2–9 in wire representations. Peptide 2 is shown in red, 3 (blue), 4 (white), 5 (pink), 6 (cyan), 7 (yellow), 8 (green) and
9 (purple). All hydrogens and side chains are omitted for clarity.
tural differences between the respective ligands, suggest-
ing some structural difference between the a-MSH
receptor and the putative yeast’s receptor.
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, col-
umn: Lichrosorb RP C18, 7 lm, 250 · 16 mm; gradient
elution: 30–100%, 70 min; mobile phase: 80% acetoni-
trile, 0.1% TFA; flow rate: 4 ml/min, 220 nm, for ana-
lytical 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: Finni-
gan TSQ 7000.
4. Experimental
4.1. Synthetic methods
Solid phase synthesis of the peptides was carried out
manually on a p-methylbenzhydrylamine resin (1 g
MBHA, 0.39 mmol/g) with standard methodology
using Boc-strategy. Side chain protecting groups were
as follows: Arg(Tos), His(Tos), Lys(2Cl-Z), Tyr(2Br-
Z). All protected amino acids were coupled in CH2Cl2
(5 ml) using DCC (2.5 equiv) and HOBt (2.5 equiv) un-
til completion (3 h) judged by Kaiser45 ninhydrin test.
After coupling of the appropriate amino acid, Boc-
deprotection was effected by the use of TFA/CH2Cl2
(1:1, 5 ml) for 5 min first then repeated for 25 min. Fol-
lowing 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 pep-
tides were cleaved from the resin with simultaneous
side chain deprotection by acidolysis with anhydrous
hydrogen fluoride (5 ml) containing 2% anisole, 8% di-
methyl sulfide and indole at 5 ꢁC for 45 min. The crude
HPLC data of the synthesized peptides:
Retention Gradient
factor
(min)
elution (%)
His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (1) 5.757
His-Phe-Arg-Trp-Gly-Lys-Phe-Val-NH2 (2) 8.786
His-Phe-Arg-Trp-Gly-Lys-Tyr-Val-NH2 (3) 7.019
His-Phe-Arg-Trp-Gly-Arg-Phe-Val-NH2 (4) 8.831
His-Phe-Lys-Trp-Gly-Lys-Phe-Val-NH2 (5) 8.524
His-Phe-Lys-Trp-Gly-Arg-Phe-Val-NH2 (6) 8.633
His-Phe-Lys-Trp-Gly-Lys-Tyr-Val-NH2 (7) 6.992
His-Tyr-Lys-Trp-Gly-Arg-Phe-Val-NH2 (8) 7.087
His-Tyr-Lys-Trp-Gly-Lys-Tyr-Val-NH2 (9) 5.173
His-Tyr-Lys-Trp-Gly-Lys-Phe-Val-NH2 (10) 6.820
His-Tyr-Arg-Trp-Gly-Lys-Phe-Val-NH2 (11) 6.947
30–55 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)
25–50 (15 min)

4356
M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
was also applied at this stage, greatly improves the
4.2. Microorganisms and media
acceptance coefficient. In this algorithm / and w angles
are changed in a manner which allows the correspond-
ing peptide group to find the most proper orientation
in the electrostatic field of the rest of the peptide chain.
The energy of the new conformation is minimized, com-
pared to the previous one and can be accepted or dis-
carded on the basis of energy and/or geometry. If the
new energy-minimized conformation is similar in shape
and in energy to the starting conformation, it is dis-
carded. Otherwise, the energy of the new conformation
is compared with the energy of the parent conformation.
If the new energy is lower, the new conformation is ac-
cepted unconditionally, otherwise the Metropolis crite-
rion53 is applied in order to accept or reject the new
conformation. If the new conformation is accepted, it re-
places the starting one; otherwise another perturbation
of the parent conformation is tried. A temperature jump
may be included if the perturbation is not successful for
an arbitrarily chosen number of iterations. The process
is iterated, until a sufficient number of conformations
have been accepted. The detailed procedure is described
in Ref. 54.
Candida spp. and Cryptococcus sp. from the American
Type Culture Collection (ATCC, Rockville, MD,
USA) were used. The panel included Candida albicans
10231, Candida tropicalis C 131 and C. neoformans
ATCC 32264. Strains were grown on Sabouraud-chlor-
amphenicol agar slants for 24 h at 35 ꢁC, maintained on
slopes of Sabouraud-dextrose agar (SDA, Oxoid). Inoc-
ula of cell suspensions were obtained according to re-
ported procedures and adjusted to 1–5 · 103 cells with
colony forming units (CFU)/mL.46
4.3. Antifungal evaluation
The test was performed in 96 wells-microplates. Peptide
test wells (PTW) were prepared with stock solutions of
each peptide in DMSO (maximum concentration
62%), diluted with RPMI-1640 to final concentrations
200 and 100 lM. Inoculum suspension (100 ll) was
added to each well (final volume in the well = 200 lL).
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., respec-
tively. Microplates were read in a VERSA Max micro-
plate 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 duplicate. Reduction of growth
for each peptide concentration was calculated as follows:
% of inhibition: 100 ꢀ (OD405 PTW ꢀ OD405SCW)/
OD405 GCW ꢀ OD405 SCW.
In order to do an extensive exploration of the conforma-
tional space, we have been carried out 10 different runs,
each of them with a different random number, for each
peptide studied. Since the EDMC procedure uses ran-
dom numbers, there is a need to initialize the random
number generator by providing an integer. Therefore,
we collected a total of 5000 accepted conformation for
each peptide studied. Each EDMC run was terminated
after 500 energy-minimized conformations had been ac-
cepted. The parameters controlling the runs were the fol-
lowing: a temperature of 298.15 K had been used for the
simulations. A temperature jump of 50,000 K had been
used; 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
conformations per iteration was 100; the fraction of ran-
dom/electrostatically-predicted conformations was 0.30;
the maximum number of steps at one increased temper-
ature was 20; and the maximum number of rejected con-
formations until a temperature jump is executed was
100. Only trans peptide bonds (x ffi 180ꢁ) had been
considered.
4.4. Statistical analysis
Data were statistically analyzed by both, one-way anal-
ysis of variance and Student’s test. A p < 0.05 was con-
sidered significant.
4.5. Computational methods
4.5.1. EDMC calculations. The conformational space of
each peptide was explored using the method previously
employed by Liwo et al.47 that included the electrostat-
ically driven Monte Carlo (EDMC) method.31,32 The
conformational energy was evaluated using the
ECEPP/3 force field.48 This force field employs rigid va-
lence geometry. The hydration energy was evaluated
using a hydration-shell model with a solvent sphere ra-
The ensemble of obtained conformations was then clus-
tered into families using the program ANALYZE,55–57
which applies the minimal-tree clustering algorithm for
separation, using backbone atoms, energy threshold of
30 kcal molꢀ1 and RMSD of 0.75 A as separation crite-
˚
ria. This procedure allows for substantial reduction of
the number of conformations and eliminates repetitions.
˚
dius of 1.4 A and atomic hydration parameters that
have optimized using non-peptide data (SRFOPT).49,50
In this model in addition to a sum of electrostatic,
non-bonding, hydrogen-bond and torsional energy
terms, the total conformational energy includes terms
accounting for loop closing and peptide solvation. The
conformation with minimized energy was subsequently
perturbed by changing its torsional / and w angles using
the Monte Carlo method.51 Piela’s algorithm,52 which
4.5.2. Molecular electrostatic potentials and molecular
interaction calculations. Quantum mechanics calcula-
tions were carried out using the Gaussian 03 program.58
We use low-energy conformations of peptide 1–11 ob-
tained from EDMC calculations. Subsequently, single
point DFT (RB3LYP/6-31G(d,p)) calculations were car-
ried out. The electronic study was carried out using

M. F. Masman et al. / Bioorg. Med. Chem. 16 (2008) 4347–4358
4357
molecular electrostatic potentials (MEP).36 These MEP
were calculated using RB3LYP/6-31G(d,p) wave func-
tions and MEP graphical presentations were created
using the MOLEKEL program.59
16. Cabiaux, V.; Agerberth, B.; Johansson, J.; Homble, F.;
Goormaghtigh, E.; Ruysschaert, J. M. Eur. J. Biochem.
1994, 224, 1019.
17. Cutuli, M.; Cristiani, S.; Lipton, J. M.; Catania, A. J.
Leukocyte Biol. 2000, 67, 233.
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19. Grieco, P.; Rossi, C.; Gatti, S.; Colombo, G.; Carlin, A.;
Novellino, E.; Lama, T.; Lipton, J. M.; Catania, A.
J. Med. Chem. 2005, 48, 1384.
Molecular graphics images were produced using the
UCSF Chimera package from the Resource for Biocom-
puting, Visualization, and Informatics at the University
of California, San Francisco (supported by NIH P41
RR-01081).60
20. Carotenuto, A.; Saviello, M. R.; Auriemma, L.; Campi-
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Acknowledgments
´
21. Kouznetsov, V. V.; Urbina, J.; Palma, A.; Lopez, S.;
Ribas, J.; Enriz, R. D.; Zacchino, S. Bioorg. Med. Chem.
2000, 8, 691.
This work is part of the Hungarian-Argentine Intergov-
ernmental S&T Cooperation Programme and was sup-
ported by the Research and Technological Innovation
Foundation as well as by grants from Universidad Nac-
ional de San Luis and grants to S.A.Z. and R.D.E. from
22. Vargas, L. Y.; Castelli, M.; Kouznetsov, V.; Urbina, J.
M.; Lopez, S.; Sortino, M.; Enriz, R. D.; Ribas, J. C.;
Zacchino, S. Bioorg. Med. Chem. 2003, 11, 1531.
23. Suvire, F. D.; Sortino, M.; Kouznetsov, V. V.; Vargas, M.
L. Y.; Zacchino, S. A.; Cruz, U. M.; Enriz, R. D. Bioorg.
Med. Chem. 2006, 14, 1851.
´
´
´
the Agencia de Promocion Cientıfica y Tecnologica de la
Argentina (ANPCyT) PICT R 260. It is part of the
Iberoamerican Program of Science and Technology for
the Development (CYTED, Network RIBIOFAR).
F.M.G. is a fellow of ANPCyT, PICT R 260. R.D.E.
is a member of the CONICET (Argentina) staff.
M.F.M. is a fellow of CONICET (Argentina).
´
24. Masman, M. F.; Rodrıguez, A. M.; Svetaz, L.; Zacchino,
S. A.; Somlai, C.; Csizmadia, I. G.; Penke, B.; Enriz, R. D.
Bioorg. Med. Chem. 2006, 14, 7604.
25. Hruby, V. J.; Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.;
Sawyer, T. K.; Staples, D. J.; de Vaux, A. E.; Dym, O.;
Castrucci, A. M.; Hintz, M. F.; Riehm, J. P.; Ranga Rao,
K. J. Med. Chem. 1987, 30, 2126.
26. Cai, M.; Mayorov, A. V.; Ying, J.; Stankova, M.; Trivedi,
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27. Fung, S.; Hruby, V. J. Curr. Opin. Chem. Biol. 2005, 9,
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28. Grieco, P.; Cai, M.; Mayorov, A. V.; Trivedi, D.; Hruby,
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.072.
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