Transcript University of Copenhagen

Peptoid analogs of the antibacterial peptide anoplin
Karin
a,b
Meinike
and Paul R.
a
Hansen
aIGM-Bioorganic
Chemistry, Faculty of Life Sciences, University of Copenhagen, Denmark
bPresent Address: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark.
Introduction
Anoplin (H-GLLKRIKTLL-NH2) is a recently discovered antibacterial peptide amide found in
the venom sac of the spider wasp Anoplius Samariensis [1]. Anoplin is active against both
Gram-positive and Gram-negative bacteria, including Staphylococcus aureus ATCC 25923 and
Eschericia coli ATCC 25922. Furthermore, the hemolytic activity against human erythrocytes is
very low.
We have previously reported a structure-activity study of anoplin [2] based on 37 analogs. The
substitutions were designed using helical wheel considerations (figure 1). We found that
substitution of Arg5 with Val, Leu, Ile, Phe,Trp or Lys resulted in analogs with MIC-values in
the range of 2-22 M. Unfortunately, the analogs were very hemolytic against human
erythrocytes.
In the present study, we have synthezised nine peptoid analogs of anoplin. In each analog, Arg5
was substituted with a peptoid monomer X (H-GLLKXIKTLL-NH2) resulting in a side chain
either identical to (Val, Leu, Ile or Phe), resembling (Trp) or different from a natural amino acid
(analogues 1-4, 5 and 6-9, respectively). Structures are shown in figure 2.
M,I,L,V
F,Y ,W
G,A,P
D,E
N,Q
K,H,R
S,T
C
Figure 1: Helical wheel of anoplin
I
6
L
10
L
3
K
7
L
2
L
9
K
4
R
5
T
8
G
1
Results and Discussion
The peptoid analogs of anoplin were synthesized using a combination of Fmoc SPPS and the submonomer approach. A purity of >95% was
obtained by preparative and analytical HPLC and the identity of the analogs was confirmed by MALDI-TOF MS.
The antibacterial activity of the analogs was tested against the Gram-positive bacterium S.aureus and the Gram-negative bacterium E.coli using a
micro-dilution assay described previously [3]. Table 1 lists the MICs obtained for the nine peptoid analogs and anoplin. Of the analogs with a
peptoid monomer yielding a side chain identical to that of a natural amino acid or resembling one, only analogs 4 and 5 showed antibacterial
activity in the concentration range used with MICs ranging from 25-100 M. The best candidates were 7-9 with MICs of 12.5-25 M against E.coli
and 25-50 M against S.aureus. Analogs 5 and 7-9 showed a clear selectivity towards E.coli, with analogs 7 and 8 having MICs comparable to those
of anoplin (23 µM and 11 µM against E.coli and S.aureus, respectively). However the selectivity was reversed.
The hemolytic activity of the analogs against human erythrocytes was determined as described previously [3]. The analogs showed very low
hemolytic activity ranging from 4 to 9 percent. The lowest hemolytic activity was observed for the analogs with the highest MICs ranging from 47%. The analogs with aromatic or ring structured side-chains had the highest hemolytic values ranging from 6 to 9 percent, though a value of 9%
still might be considered fairly low.
Table: Minimum inibitory concentration (M) and hemolytic activity (50 M) of the analogs
Sequence
anoplin
1
[N-(2-propyl)Gly5] anoplin
2
[N-(2-methylpropyl)Gly5]anoplin
3
[N-(1-methylpropyl)Gly5]anoplin
4
[N-(benzyl)Gly5]anoplin
5
[N-(2-(1H-Indol-3-yl)ethyl)Gly5]anoplin
6
[N-(butyl)Gly5]anoplin
7
[N-(2,2-diphenylethyl)Gly5]anoplin
8
[N-(1-naphthalenemethyl)Gly5]anoplin
9
[N-(cyclohexylmethyl)Gly5]anoplin
S.aureus
E.coli
11
23
-
>100
>100
4
>100
>100
7
>100
>100
4
>100
50-100
6
50-100
25
8
>100
100
5
25
12.5
7
25
12.5
9
50
25
9
Figure 2: Peptoid building blocks used in this study
% hemolysis
Conclusion
In conclusion, we have shown that Arg5 of anoplin may be substituted with either N-(2,2-diphenylethyl)Gly or N-(1-naphthalenemethyl)Gly,
resulting in peptoid analogs with a hemolytic activity and antibacterial activity which are comparable to anoplin, however with reversed selectivity.
Since peptoid residues are stable to proteolytic breakdown, this study indicates that peptoid analogs of anoplin are promising lead-structures for
the development of new antibacterial agents.
Acknowledgements
We thank Ms. Jette Petersen for excellent technical assistance. This work was
supported by the Danish Research Council grant number 9900234, the Augustinus
Foundation, the Frimodt–Heineke Foundation, the Family Hede Nielsen
Foundation, the JS Foundation and the Aase & Ejnar Danielsens Foundation.
References
1. Konno, K. et al. Biochim Biophys Acta, 2001, 1550, 70-80.
2. Ifrah, D., Doisy, X., Ryge, T.S., and Hansen, P.R. J Pept Sci., 2005, 11,113-21.
3. Nielsen, S.L., Frimodt-Møller, N., Kragelund B.B. and Hansen, P.R. Protein Science, 2007, 16, 1969-1976.