Dissociation of Antibacterial and Hemolytic Activities of an Amphipathic Peptide Antibiotic

Article abstract of DOI:10.1021/jm0341352

Using an alanine-scanning method, we have found that the antibacterial and hemolytic activities of the amphipathic cyclic decapeptide antibiotic tyrocidine A depend on different structural components. Single substitution of glutamine-6 of the natural product with a cationic amino acid results in a therapeutic index enhancement of up to 140-fold. Successful dissociation of the two intimately associated properties should enable discovery of novel analogues with both high bacterial selectivity and antibacterial potency to counter microbial resistance.

Full text of DOI:10.1021/jm0341352

4
830
J . Med. Chem. 2003, 46, 4830-4833
Dissocia tion of An tiba cter ia l a n d
Hem olytic Activities of a n Am p h ip a th ic
P ep tid e An tibiotic
Sch em e 1. Synthesis of Tyrocidine A and Its
Alanine-Substituted Analogues
a
Chuanguang Qin, Xiaofen Zhong, Xianzhang Bu,
Na Lee J oyce Ng, and Zhihong Guo*
Department of Chemistry and Biotechnology Research
Institute, The Hong Kong University of Science and
Technology, Clear Water Bay, Kowloon,
Hong Kong SAR, China
Received J uly 1, 2003
Abstr a ct: Using an alanine-scanning method, we have found
that the antibacterial and hemolytic activities of the amphi-
pathic cyclic decapeptide antibiotic tyrocidine A depend on
different structural components. Single substitution of glut-
amine-6 of the natural product with a cationic amino acid
results in a therapeutic index enhancement of up to 140-fold.
Successful dissociation of the two intimately associated prop-
erties should enable discovery of novel analogues with both
high bacterial selectivity and antibacterial potency to counter
microbial resistance.
In tr odu ction . Microbial resistance to currently avail-
able antibiotics has become a serious public health
1
threat. Among efforts to contain the resistance, interest
has been focused on the antibacterial R-helical peptides,
including melittin2 and pardaxin,3 and amphipathic
â-sheet peptide antibiotics such as gramicidin S and
a
Reagents and conditions: (a) standard Fmoc solid-phase
peptide synthesis; (b) ICH2CN, NMP, DIPEA, 24 h; (c) CF3COOH/
phenol/i-Pr SiH/H O ) 88:5:5:2, 1 h; (d) 20% DIPEA/THF.
3
2
4
tyrocidine A. These natural products act on microbial
by us as shown in Scheme 1,10 based on the propensity
of the biosynthetic precursor of tyrocidine A to form a
conformation highly favorable for head-to-tail cycliza-
cell membranes where their accumulation results in
5
disruption of the barrier functions. Development of
resistance to such molecules is unlikely because it
requires significant alteration of the lipid composition
of the microbial membranes. Indeed, no resistance to
1
1
tion. Briefly, the linear precursors of the cyclic pep-
tides were synthesized on 4-sufamylbutyryl AM resin
using standard Fmoc/DIC/HOBt chemistry. The carboxy
terminus of the linear peptide was activated by cyano-
methylation with ICH2CN. After deprotection of the Boc
and t-Bu groups, the activated linear precursors were
cyclized under basic conditions to afford specific head-
to-tail cyclic products, without interference of the reac-
tive side chain -NH2 and -OH groups. The products
were purified by HPLC and characterized by H NMR
and FAB-MS before determination of the biological
activities.
Resu lts a n d Discu ssion . To identify the structural
determinants for the antibacterial and hemolytic activi-
ties, constituent amino acid residuals were systemati-
cally substituted with alanine in an alanine-scanning
experiment. The alanine analogues were subjected to
determination of the minimum inhibition concentration
(MIC) against Bacillus subtilis and minimum hemolysis
6
such antibiotics has been reported. However, it is well-
known that the high antibacterial potency of these
amphipathic peptides is often intimately associated with
high hemolytic activity, preventing their direct use in
combating the microbial resistance.
To develop drug leads against resistant microbes on
the basis of these natural peptide scaffolds, the struc-
ture-activity relationship was exploited to increase the
therapeutic index, namely, minimizing the high hemolyt-
ic activity while maintaining the original high antibac-
terial activity. For example, D-amino acids have been
1
7
8
systematically introduced to the R-helical and â-sheet
peptides to disrupt the amphipathic structures for
enhancement of selectivity against bacteria. In addition,
chemoenzymatic replacement of a hydrophobic resident
of tyrocidine A with a cationic one results in significant
9
increase in microbial specificity. To further unravel the
concentration (MHC) of human erythrocytes using
structure-activity relationship for the small peptide
antibiotic tyrocidine A, we took a systemic approach to
identify the structural determinants of its antibacterial
and hemolytic properties that led to dissociation of the
two closely associated properties.
published methods with slight modifications.1
2,13
The
results are summarized in Table 1. The relative thera-
peutic indices (relative MHC/MIC) of the analogues to
the wild-type tyrocidine A are plotted in Figure 1.
Table 1 and Figure 1 indicate that most side chains
of the constituent amino acid residuals affect the
antibacterial and hemolytic activities to approximately
the same extent. Alanine substitution of D-Phe-1, Tyr-
7, Val-8, or Orn-9 resulted in analogues with therapeutic
Syn th esis. Tyrocidine A and its analogues were
synthesized by a safety-catch method developed earlier
*
To whom correspondence should be addressed. Phone: 852-2358-
7
352. Fax: 852-2358-1594. E-mail: chguo@ust.hk.
1
0.1021/jm0341352 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/02/2003

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4831
Ta ble 1. Antibacterial and Hemolytic Activities of Tyrocidine
A and Its Alanine-Substituted Analogues
substituted
residual
MHCa
(µg/mL)
MICb
(µg/mL)
compd
MHC/MIC
1
2
3
4
5
6
7
8
9
none
8.2 ( 2.0
50 ( 5
13 ( 2
16 ( 3
14 ( 2
28 ( 2
39 ( 3
47 ( 4
47 ( 4
53 ( 5
20 ( 3
8.3 ( 1.2
25 ( 3
13 ( 1
15 ( 2
14 ( 1
1.5 ( 0.5
15 ( 1
10 ( 1
19 ( 2
18 ( 1
15 ( 1
1.0 ( 0.4
2.0 ( 0.5
1.0 ( 0.3
1.1 ( 0.3
1.0 ( 0.2
19 ( 6
2.6 ( 0.4
4.7 ( 0.8
2.5 ( 0.4
2.9 ( 0.5
1.3 ( 0.3
1
0
Leu
Orn
9
8
Val
Tyr
Gln
Asn
7
6
5
4
1
D-Phe
3
Phe
Pro
2
1
1
0
1
F igu r e 2. Tyrocidine A analogues displaying various side
chain functionalities at the position of Gln-6.
D-Phe
a
Minimum hemolysis concentration determined with fresh
human erythrocytes. b Minimum inhibition concentration deter-
mined with Bacillus subtilis (ATCC 82).
Ta ble 2. Antibacterial and Hemolytic Activities of Tyrocidine
A Analogues with Various Side Chains at the Position of Gln-6
amino acid at
position-6
MHCa
(µg/mL)
MICb
(µg/mL)
compd
MHC/MIC
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
Leu
Val
Ile
20 ( 2
35 ( 2
45 ( 4
18 ( 2
125 ( 8
120 ( 6
25 ( 3
99 ( 5
42 ( 3
16 ( 2
32 ( 2
30 ( 2
20 ( 2
0.3 ( 0.1
2.9 ( 0.5
1.8 ( 0.3
0.6 ( 0.1
3.8 ( 0.3
5.0 ( 0.5
0.2 ( 0.1
1.1 ( 0.2
0.3 ( 0.1
0.3 ( 0.1
5.3 ( 0.7
7.5 ( 0.5
0.4 ( 0.2
67 ( 12
12 ( 3
25 ( 5
Phe
Trp
Tyr
Orn
Lys
Arg
Ser
Asp
Glu
Asn
30 ( 8
33 ( 4
24 ( 4
125 ( 35
90 ( 22
140 ( 43
53 ( 18
6.0 ( 1.2
4.0 ( 0.5
50 ( 20
pronounced effect of alanine substitution at the Gln-6
side chain position as shown in Table 1 and Figure 1
has not been reported before. Substitution of the polar
side chain amide on this residual by a small methyl
group led to substantial weakening of the hemolytic
activity with concurrent significant enhancement of
antibiotic potency of the resulting cyclic peptide. Both
effects of such a structural modification are beneficial
to identification of novel antibiotic candidates without
cytotoxic activity.
F igu r e 1. Determination of effects of amino acid side chains
on the therapeutic index of tyrocidine A by alanine scanning.
indices essentially equal to that of the wild type natural
product, with modest loss of antibacterial potency. This
indicates that modification of these residuals is unlikely
to improve the antibacterial selectivity of the resulting
cyclic peptides. On the other hand, substitution of
residuals Pro-2, Phe-3, Asn-5, and Leu-10 by alanine
brings about modest improvement of the MHC/MIC
ratio (2- to 3-fold). However, the improvement of the
selectivity against bacteria is accompanied by a decrease
in the antibacterial activity by 2- to 3-fold, indicating
that the modification provides modest differentiation of
the two biological properties of the cyclic peptides with
sacrifice of the antibiotic potency. These observations
show that the biological activities of the cyclic peptides
are indeed intimately associated and mostly dependent
on the structure, consistent with previous observa-
To further exploit the potential of the sensitivity of
the biological activities to the residual Gln-6, various
side chain functionalities were individually displayed
at this position to examine their effects on the activities
of the resulting cyclic peptide products. Synthesis of
these tyrocidine A analogues (Figure 2) was accom-
plished with the method illustrated in Scheme 1. After
HPLC purification and characterization of the cyclic
peptide products, they were subjected to the same
antibacterial and hemolytic activity determination.
As shown in Table 2 and Figure 3, significant further
increase in therapeutic index is achieved when the
cationic amino acid residual Orn, Lys, or Arg is intro-
duced into position 6, in comparison to the alanine-
substituted analogue at the same position. The enhance-
ment is mainly due to the increase of antibiotic potency.
In contrast, incorporation of the anionic side chain of
Asp or Glu results in a decrease in the MHC/MIC ratio.
When the alanine is replaced by other small aliphatic
amino acids, further enhancement of the therapeutic
index was only observed for the leucine- and serine-
substituted analogues. The branched hydrophobic ali-
phatic side chain of valine or isoleucine does not increase
the bacterial selectivity of the resulting analogues nor
do the aromatic side chain functionalities of phenyla-
4
,8,12
tions.
Of particular interest are the results of the alanine
substitution at Gln-6 and D-Phe-4, which show signifi-
cant differentiation of the antibacterial and hemolytic
activities with an MHC/MIC of 19 and 4.7, respectively.
Most importantly, the significant increase of the thera-
peutic index is achieved without affecting or increasing
the antibacterial activity, showing a complete segrega-
tion of the two intimately correlated biological proper-
ties. Increase of the therapeutic index for the alanine
substitution at D-Phe-4 is consistent with the results of
a previous investigation in which an even higher MHC/
MIC ratio was achieved when the hydrophobic side
chain was changed to a cationic one such as D-Arg,
D-Lys, or D-Orn.9 On the other hand, an even more

4
832 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Letters
side chain. However, the contribution of the Gln-6 side
chain to the amphipathicity of the cyclic peptide is less
certain because it is not obvious whether it belongs to
the hydrophobic or hydrophilic side of the scaffold
molecule. Substitution at both D-Phe-4 and Gln-6 is less
likely to exert its effect on the biological activities
through conformational change because the rigid anti-
parallel â-pleated sheet structure of the cyclic peptides
is maintained by four strong interstrand hydrogen
bonds that are not susceptible to side chain variations.
A significant increase in the therapeutic index was
reported when a cationic side chain functionality was
introduced into the position of D-Phe-4. This was
ascribed to the differential ionic interactions between
the analogues and charged headgroups of lipid compo-
F igu r e 3. Effect of side chain functionalities at the position
of Gln-6 on the therapeutic index.
9
nents of the membranes. Similar differential ionic
interaction should also be responsible for the increase
in therapeutic index when positively charged side chains
are introduced into the position of Gln-6 in the current
study because the additional positive charge will greatly
strengthen binding affinity of the resulting analogue for
the prokaryotic membrane that contains predominantly
negatively charged phospholipids, whereas its effect on
the analogue’s affinity for eukaryotic membrane will be
much less significant because of the zwitterionic nature
of the phospholipids in mammalian cells. However,
effects of the additional positive charge at the positions
of D-Phe-4 and Gln-6 are different. Substitution of the
former residual with a basic amino acid results in a
significant increase of MHC without much effect on
F igu r e 4. Antiparallel â-pleated sheet structure of tyrocidine
A.
laine, tryptophan, and tyrosine. These results indicate
that the antibacterial potency and the bacterial selectiv-
ity can be significantly increased when a cationic or
unbranched small aliphatic amino acid is incorporated
into the position of Gln-6 of the wild-type tyrocidine A
template. Notably, analogues with high therapeutic
indices also exhibit significant increase in activity
against drug-resistant microbes.14
It is interesting to note that the hemolytic and
antibiotic properties of the cyclic peptide natural product
depend on different structural components. The poten-
tially useful effects of modifications at D-Phe-4 and
Gln-6 come from the unique structure and mode of
action of the cyclic peptide scaffold molecule. Tyrocidine
A is an amphipathic peptide antibiotic with one hydro-
phobic side consisting of Phe-2, Asn-5, Val-8, and Leu-
9
MIC, whereas the increase in the therapeutic index
caused by the same substitution at the latter position
is mostly due to the enhancement of the antibacterial
potency (decrease in MIC) with a concurrent moderate
increase in MHC. Interestingly, substitution with small
aliphatic amino acids at Gln-6 also results mainly in a
decrease of MIC. These different effects indicate that
analogues with high antibacterial specificity and po-
tency are accessible by simultaneous structural modi-
fication at both positions.
Con clu sion . We have discovered a novel position on
the scaffold of natural cyclic decapeptide antibiotic
tyrocidine A at Gln-6 whose modification will signifi-
cantly lower the unwanted hemolytic activity and
simultaneously enhance the desired antibacterial activ-
ity, in addition to our confirmation of similar effects of
structural variation at another position of D-Phe-4 in a
previous report. Combination of the favorable structural
variations at both positions should enable identification
of analogues of the natural product that are capable of
combating microbial resistance without provoking new
resistance.
1
0 and a hydrophilic cationic Orn-9 on the other side of
the rigid antiparallel â-pleated sheet structure (Figure
1
5
4
). Previous studies on R-helical and â-sheet peptide
antibiotics showed that the amphipathicity often affects
the antibiotic potency and the hemolytic activity in
4
b,8,12
parallel.
Being on the same side as the hydrophobic
patch of tyrocidine A, the aromatic side chain of D-Phe-4
contributes to the amphipathicity of the antibiotic.
Increase of antibiotic potency and concurrent decrease
of the hemolytic activity of the cyclic peptide by substi-
tuting this residual with alanine indicate that the
aromatic side chain is detrimental to the interaction
with mammalian cell membrane while facilitating its
binding with the bacterial membrane, showing a dif-
ferentiating amphipathicity effect on the two biological
activities of the cyclic analogue. A similar differentiating
effect of the polar side chain of Gln-6 may also underlie
the dissociation of antibiotic and hemolytic activities
caused by its substitution with an alanine or leucine
Ack n ow led gm en t. This work was supported in part
by the Innovation and Technology Fund (Grant ITS/119/
0) and the RGC (Grant DAG01/02.SC09) from the
Government of the Hong Kong Special Administrative
Region and from HKUST (Grant HIA98/99.SC03). We
also thank the Department of Chemistry at the HKUST
for providing postgraduate studentships to N. L. J . Ng.
0
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and HPLC, MS, and 1H NMR data for 1-24. This
material is available free of charge via the Internet at http://
pubs.acs.org.

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4833
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