Synthetic mimics of antimicrobial peptides with immunomodulatory responses

Article abstract of DOI:10.1021/ja303304j

A new series of aryl-based synthetic mimics of antimicrobial peptides (SMAMPs) with antimicrobial activity and selectivity have been developed via systematic tuning of the aromatic groups and charge. The addition of a pendant aromatic group improved the a

Full text of DOI:10.1021/ja303304j

Communication
pubs.acs.org/JACS
Synthetic Mimics of Antimicrobial Peptides with Immunomodulatory
Responses
Hitesh D. Thaker,†,∥ Abhigyan Som,†,‡,∥ Furkan Ayaz,‡ Dahui Lui,§ Wenxi Pan,§ Richard W. Scott,§
Juan Anguita,*,‡ and Gregory N. Tew*,†,‡
†
‡
Departments of Polymer Science and Engineering and Veterinary & Animal Sciences, University of Massachusetts, Amherst,
Massachusetts 01003, United States
§PolyMedix Inc., 170 North Radnor-Chester Road, Suite 300, Radnor, Pennsylvania 19087, United States
S
* Supporting Information
to reproduce critical AMP biophysical characteristics such as
cationic charge and amphiphilic structure.
ABSTRACT: A new series of aryl-based synthetic mimics
of antimicrobial peptides (SMAMPs) with antimicrobial
activity and selectivity have been developed via systematic
tuning of the aromatic groups and charge. The addition of
a pendant aromatic group improved the antimicrobial
activity against Gram-negative bacteria, while the addition
of charge improved the selectivity. SMAMP 4 with six
charges and a naphthalene central ring demonstrated a
selectivity of 200 against both Staphylococcus aureus and
Escherichia coli, compared with a selectivity of 8 for the
peptide MSI-78. In addition to the direct antimicrobial
activity, SMAMP 4 exhibited specific immunomodulatory
activities in macrophages both in the presence and in the
absence of lipopolysaccharide, a TLR agonist. SMAMP 4
also induced the production of a neutrophil chemo-
attractant, murine KC, in mouse primary cells. This is the
first nonpeptidic SMAMP demonstrating both good
antimicrobial and immunomodulatory activities.
Systematic structure−activity relationship (SAR) studies on
SMAMPs have led to the development of several compounds
that exhibit direct antimicrobial activity and reduced cytotox-
icity, some of which are already in clinical development.20
However, greater potential may lie in harnessing the innate
immune system to combat bacterial infections.21 Toward this
end, a number of immunomodulatory molecules, including
synthetic HDP mimics and innate defense regulators (IDRs)
that do not show direct antimicrobial response, are being
developed into therapeutic candidates and adjuvants.22 The
combination of direct antimicrobial activity and controlled
immunomodulation in an antimicrobial system would present a
novel strategy for treating infections with multiple mechanisms
of action against pathogens, thus minimizing pathogen−
antimicrobial responses. Herein we report a new series of
aryl-based SMAMPs with improved Gram-negative and Gram-
positive antimicrobial activities, some of which have signifi-
cantly enhanced selectivity and reduced toxicity compared with
the well-known magainin analogue MSI-78, which is in phase-
III clinical trials as a topical antibiotic.20 One of these new
SMAMPs also showed unique immunomodulatory responses.
We previously reported a new series of SMAMPs based on
simple aryl scaffolds synthesized via Suzuki coupling.23 The use
of Suzuki coupling was advantageous because of its mild
reaction conditions, high functional group tolerance, and easy
scalability. However, the design of these SMAMPs, with only
three aryl rings and two positive charges, proved to be
insufficient to achieve antimicrobial activity and selectivity
comparable to those of the previously described oligomeric
SMAMPs.
he rise in bacterial resistance and the declining approval
T
rate of novel anti-infective drugs are a major threat to
global public health, especially in hospitals and other health
care settings.1 Antimicrobial peptides (AMPs), which are found
in almost every multicellular organism, have attracted
considerable attention as models for the design of new
therapeutic agents because of their broad-spectrum activity
and reduced bacterial resistance development.2 AMPs form the
core of the innate immune system that effectively deals with
microbial invasion, and most AMPs show direct antimicrobial
activity against a variety of pathogens. Only recently has it been
recognized that they play a key role in immunomodulation and
hence have also been termed host defense peptides (HDPs).3
For example, LL-37 and defensins affect innate immune cell
functions, including the induction and modulation of chemo-
kine and cytokine production, direct chemoattraction of
immune cells, angiogenesis promotion, and wound healing.
Despite the many interesting properties of these natural
peptides, their development into therapeutic agents has been
limited because of their toxicity, high manufacturing cost, and
poor in vivo efficacy.3 This has driven research toward the
development of non-natural AMPs or synthetic mimics of
AMPs (SMAMPs), including non-natural peptide mimics4−7 as
well as synthetic polymers8−17 and oligomers,17−19 which aim
In this work, a new series of aryl SMAMPs was designed in
order to evaluate the effect of charge and aromatic group
hydrophobicity on the biological activity while still maintaining
an amphiphilic topology. Four and six cationic charges were
investigated, and three different central rings were used to tune
the overall hydrophobicity: benzene, naphthalene, and phenyl-
benzene [see the Supporting Information (SI) for the synthesis
and characterization of the SMAMPs]. The hydrophobicities of
the aryl SMAMPs were quantified using reversed-phase HPLC
Received: April 11, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja303304j | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Table 1. Antibacterial Activities and Selectivities of SMAMPs
a
b
Measured by HPLC using a C8 column with a gradient of 1% acetonitrile/min starting with 100% water. Data from ref 23.
retention times (Rt). The antimicrobial activities [expressed as
minimum inhibitory concentration (MIC)] of these SMAMPs
were tested against four pathogens, including both Gram-
negative and Gram-positive bacteria, and their hemolysis
[evaluated in terms of HC50, the lowest concentration that
causes 50% hemolysis of red blood cells (RBCs)] was tested
against human RBCs (Tables S1 and S2 in the SI).
potency was also increased (MIC ≈ 3 μg/mL vs 16 μg/mL for
MSI-78). SMAMP 5 did not show a significant improvement in
the antimicrobial activity relative to SMAMP 3, which was
already active, but it had better selectivity for S. aureus. Similar
results have been observed for antimicrobial polynorbornenes,
where increasing the charge density led to nonhemolytic and
active polymers.25 These data confirm that increasing the
charge improves the selectivity.
Table 1 summarizes the biological activities of the SMAMPs
in comparison with MSI-78. Relative to the previously studied
triaryl benzene oligomers,23 SMAMP 1 containing a benzene
central ring showed a significant decrease in hemolytic activity
(HC50 > 1000 μg/mL vs 36 μg/mL), although the
antimicrobial activity was still low. The reduced toxicity was
attributed to the increase in hydrophilicity due to the greater
number of cationic charges (4 vs 2).23 Changing the central
ring from benzene (SMAMP 1) to naphthalene (SMAMP 2)
increased the hydrophobicity but did not alter the antimicrobial
activity. SMAMP 2 was more hemolytic and thus had a lower
selectivity than SMAMP 1. SMAMP 3 with a pendant phenyl
group was the most hydrophobic among the three compounds
in the series containing four charges (Rt = 28.8 min). Relative
to SMAMP 2, SMAMP 3 showed an 8-fold increase in
antimicrobial activity against Escherichia coli (MIC = 3.13 μg/
mL) as well as a higher HC50 and thus an improved selectivity
of 172. The increased activity against E. coli for SMAMP 3 was
attributed to the arrangement of the hydrophobic pendant
aromatic ring, which is known to insert into the membrane
interface.24
To elucidate the role of charge, SMAMPs 4 and 5, each with
six charges, were designed and synthesized for comparison to
their analogues with four charges (SMAMPs 2 and 3,
respectively). SMAMPs 4 and 5 containing naphthalene and
phenylbenzene central rings, respectively, had higher HC50
values (i.e., were less toxic against RBCs) than SMAMPs 2
and 3. SMAMP 4 showed improved antimicrobial activity
against both Staphylococcus aureus and E. coli relative to
SMAMP 2, resulting in a very high selectivity of 200. This was
almost 20 times higher than the selectivity of MSI-78, and the
In addition to the direct antimicrobial activity demonstrated
by SMAMPs 2−5, their ability to modulate the immune
response was measured in terms of the production of the pro-
inflammatory cytokines tumor necrosis factor (TNF) and
interleukin-6 (IL-6) and the anti-inflammatory cytokine
interleukin-10 (IL-10) in the murine macrophage cell line
RAW 264.7. Among the antimicrobial SMAMPs reported here,
only SMAMP 4 induced TNF production in RAW 264.7 cells
(Figure 1A). Natural peptides such as human neutrophil α-
defensins are known to stimulate TNF production in
monocytes.26 Lipopolysaccharide (LPS),27 a bacterial cell wall
component, and bacterial DNA (CpG-ODN)28 are also known
for their direct stimulation of TNF in macrophages, and some
synthetic analogues such as polysaccharides (branched
chitins)29 and monophosphoryl lipid A (MLA) have been
reported to induce TNF production.30 However, to the best of
our knowledge, no SMAMP has been reported to have
agonistic (immunostimulatory) activity. LPS contamination
during SMAMP 4 preparation was ruled out, since pretreat-
ment of RAW 264.7 cells with polymyxin B, known for its high
LPS binding affinity,27 did not affect the ability of SMAMP 4 to
increase TNF production (Figure S6 in the SI). In addition, the
TNF production varied with the SMAMP concentration,
indicating controlled stimulation by SMAMP 4. To evaluate
the immunomodulatory effect of these SMAMPs in the
presence of LPS, RAW 264.7 cells were preincubated with
the SMAMPs and then stimulated with LPS (Figure S7).
SMAMP 4 increased the LPS-induced production of the pro-
inflammatory cytokines TNF and IL-6 (Figure 1B,C) but
inhibited the production of the anti-inflammatory cytokine IL-
B
dx.doi.org/10.1021/ja303304j | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 2. RAW 264.7 cells were preincubated with or without mouse
recombinant IL-10 (50 ng/mL) and then with SMAMP 4 (5.0 μg/
mL) or 0.05% DMSO for 1 h, followed by stimulation with or without
LPS (100 ng/mL) for 18 h. The supernatants were analyzed for TNF.
The data are presented as mean sem of triplicate samples (***, P <
0.001; ns, nonsignificant; the means were compared using Student’s t
test).
Figure 1. RAW 264.7 cells were preincubated with SMAMP (5.0 μg/
mL) or 0.05% DMSO for 1 h and stimulated with or without LPS
(100 ng/mL) for 18 h. The supernatants were analyzed by ELISA for
(A, B) TNF, (C) IL-6, and (D) IL-10 production. The data are
presented as mean standard error of the mean (sem) for triplicate
samples and are representative of three independent experiments (*, P
< 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant; the means
were compared using Student’s t test).
10 to background levels (Figure 1D). This unique ability of
SMAMP 4 distinguishes it from peptides such as LL-37 and
IDR peptides, which do not directly stimulate TNF production
but do suppress LPS-induced TNF production by upregulation
of IL-10 and, in the case of LL-37, also by binding to LPS.31,−33
On the basis of the results shown in Figure 1A, it was not
surprising that SMAMPs 2, 3, and 5 did not cause any
additional increase in TNF production upon LPS stimulation,
although SMAMP 3 showed a marginal decrease in LPS-
induced TNF production (Figure S7).
Figure 3. Mouse BMDM cells were preincubated with SMAMP 4 (5.0
μg/mL) or 0.05% DMSO for 1 h and stimulated with or without LPS
(100 ng/mL) for 15 h. The supernatants were analyzed for (A) TNF,
(B) IL-10, and (C) murine KC. The data are presented as the mean
sem of triplicate samples and are representative of two to three
independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001;
the means were compared using Student’s t test).
The anti-inflammatory cytokine IL-10 itself is known to
inhibit LPS-induced TNF production in RAW cells.34,35 Thus,
to evaluate the possible correlation, if any, between the pro- and
anti-inflammatory cytokine release activities, the ability of
SMAMP 4 to enhance LPS-induced TNF production in RAW
cells preincubated with externally added mouse recombinant
IL-10 (rIL-10) was investigated. Figure 2 shows that the
addition of rIL-10 did not significantly affect the SMAMP 4-
mediated self-agonistic effect in RAW 264.7 cells, as only a
slight decrease in the overall TNF level was observed. However,
upon LPS stimulation, the presence of rIL-10 resulted in the
abrogation of SMAMP 4’s capacity to enhance TNF
production. This observation suggests that in LPS-stimulated
RAW 264.7 cells, the SMAMP 4-mediated decrease in IL-10
production was at least partially responsible for increased TNF
production. Therefore, SMAMP 4 seems to orchestrate a
balance of the pro- and anti- inflammatory cytokine responses
in macrophages. Many fundamental questions still remain, but
these unique immunomodulatory properties of SMAMP 4 can
be used to trigger immune responses in a very specific way. For
example, MLA, a toll-like receptor 4 (TLR4) agonist that
induces enhanced TNF production similar to SMAMP 4, is
already an effective adjuvant for hepatitis B and influenza.22
The specific self-agonistic effect of SMAMP 4 along with its
elevated agonistic effect with LPS stimulation in RAW 264.7
cells prompted us to evaluate the SMAMP 4 activity in primary
murine bone marrow-derived macrophages (BMDMs). In the
absence of LPS, SMAMP 4 induced a small amount of TNF
production (30 pg/mL) in BMDMs (Figure 3A); however,
upon LPS stimulation, SMAMP 4 significantly increased the
LPS-induced TNF production and inhibited the LPS-induced
IL-10 production (Figure 3A,B). This observation is very
similar to the immunomodulatory effect of SMAMP 4 in RAW
264.7 cells.
Besides its ability to modulate pro- and anti-inflammatory
cytokine production in RAW 264.7 and BMDM cells, SMAMP
4 also induced significantly higher levels of murine KC
(chemokine CXCL1, a neutrophil chemoattractant) relative
to the DMSO control in BMDMs (Figure 3C). Increased KC
expression has been found to be associated with neutrophil
influx in a range of inflammatory conditions.36 It was previously
reported that the protective activity of a synthetic cationic
peptide against bacterial infection was associated with the
induction of chemokines such as CXCL1 from macrophages
and/or monocytes.37 Thus, the ability of this nontoxic,
nonpeptidic, antimicrobial SMAMP to modulate both cytokine
and chemokine production is encouraging for the design of
synthetic molecules with multiple biological functions.
C
dx.doi.org/10.1021/ja303304j | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(16) Sambhy, V.; Peterson, B. R.; Sen, A. Angew. Chem. Int. Ed. 2008,
47, 1250.
(17) Choi, S.; Isaacs, A.; Clements, D.; Liu, D.; Kim, H.; Scott, R. W.;
Winkler, J. D.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
6968.
(18) Tang, H.; Doerksen, R.; Jones, T.; Klein, M.; Tew, G. Chem.
Biol. 2006, 13, 427.
(19) Tew, G. N.; Clements, D.; Tang, H.; Arnt, L.; Scott, R. W.
Biochim. Biophys. Acta 2006, 1758, 1387.
(20) Fjell, C. D.; Hiss, J. A.; Hancock, R. E. W.; Schneider, G. Nat.
Rev. Drug Discovery 2012, 11, 37.
(21) Finlay, B. B.; Hancock, R. E. Nat. Rev. Microbiol. 2004, 2, 497.
(22) Nicholls, E. F.; Madera, L.; Hancock, R. E. Ann. N.Y. Acad. Sci.
2010, 1213, 46.
(23) Thaker, H. D.; Sgolastra, F.; Clements, D.; Scott, R. W.; Tew, G.
N. J. Med. Chem. 2011, 54, 2241.
(24) White, S. H.; Wimley, W. C. Annu. Rev. Biophys. Biomol. Struct.
1999, 28, 319.
(25) Al-Badri, Z. M.; Som, A.; Lyon, S.; Nelson, C. F.; Nusslein, K.;
The new series of aryl-based SMAMPs described here, which
were designed via systematic tuning of hydrophobicity and
cationic charge, exhibited potent antibacterial activities relative
to MSI-78 while being nontoxic to host cells. Additionally,
SMAMP 4 exhibited unique immunomodulatory properties.
The dual-functional role of SMAMPs with direct antimicrobial
activity and immunomodulatory response is very encouraging
since immunomodulatory compounds have gained importance
in recent years in anti-infective therapy, cancer therapy, and
vaccine development. These SMAMPs were originally designed
to execute antimicrobial activity, but their ability to boost the
innate immune response represents a promising approach to
prevent or treat infectious diseases.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details, HPLC data, broad-spectrum antimicrobial
activity, and additional figures. This material is available free of
charge via the Internet at http://pubs.acs.org.
Tew, G. N. Biomacromolecules 2008, 9, 2805.
(26) Chaly, Y. V.; Paleolog, E. M.; Kolesnikova, T. S.; Tikhonov, I. I.;
Petratchenko, E. V.; Voitenok, N. N. Eur. Cytokine Network 2000, 11,
257.
(27) Gough, M.; Hancock, R. E.; Kelly, N. M. Infect. Immun. 1996,
64, 4922.
(28) Lipford, G. B.; Sparwasser, T.; Bauer, M.; Zimmermann, S.;
Koch, E. S.; Heeg, K.; Wagner, H. Eur. J. Immunol. 1997, 27, 3420.
(29) Shimojoh, M.; Kojima, T.; Nakajima, K.; Hatta, K.; Katoh, A.;
Kurita, K. Biomacromolecules 2010, 11, 1212.
(30) Henricson, B. E.; Manthey, C. L.; Perera, P. Y.; Hamilton, T. A.;
Vogel, S. N. Infect. Immun. 1993, 61, 2325.
AUTHOR INFORMATION
Corresponding Author
janguita@vasci.umass.edu; tew@mail.pse.umass.edu
■
Author Contributions
∥H.D.T. and A.S. contributed equally.
Notes
The authors declare no competing financial interest.
(31) Scott, M. G.; Dullaghan, E.; Mookherjee, N.; Glavas, N.;
Waldbrook, M.; Thompson, A.; Wang, A.; Lee, K.; Doria, S.; Hamill,
P.; Yu, J. J.; Li, Y.; Donini, O.; Guarna, M. M.; Finlay, B. B.; North, J.
R.; Hancock, R. E. Nat. Biotechnol. 2007, 25, 465.
(32) Mookherjee, N.; Hamill, P.; Gardy, J.; Blimkie, D.; Falsafi, R.;
Chikatamarla, A.; Arenillas, D. J.; Doria, S.; Kollmann, T. R.; Hancock,
R. E. Mol. BioSyst. 2009, 5, 483.
(33) Rosenfeld, Y.; Papo, N.; Shai, Y. J. Biol. Chem. 2006, 281, 1636.
(34) Dagvadorj, J.; Naiki, Y.; Tumurkhuu, G.; Hassan, F.; Islam, S.;
Koide, N.; Mori, I.; Yoshida, T.; Yokochi, T. Innate Immun. 2008, 14,
109.
(35) Clarke, C. J. P.; Hales, A.; Hunt, A.; Foxwell, B. M. J. Eur. J.
Immunol. 1998, 28, 1719.
(36) Lira, S. A.; Zalamea, P.; Heinrich, J. N.; Fuentes, M. E.;
Carrasco, D.; Lewin, A. C.; Barton, D. S.; Durham, S.; Bravo, R. J. Exp.
Med. 1994, 180, 2039.
ACKNOWLEDGMENTS
■
This work was supported by NIH (AI-074866 and U01 AI-
082192). The authors acknowledge Ms. Melissa Lackey and Dr.
Federica Sgolastra for their invaluable comments on the early
drafts. Mass spectral data were obtained at the University of
Massachusetts Mass Spectrometry Facility, which is supported
in part by NSF.
REFERENCES
(1) Taubes, G. Science 2008, 321, 356.
(2) Zasloff, M. Nature 2002, 415, 389.
■
(3) Hancock, R. E. W.; Sahl, H.-G. Nat. Biotechnol. 2006, 24, 1551.
(4) Park, Y.; Lee, D. G.; Jang, S. H.; Woo, E. R.; Jeong, H. G.; Choi,
C. H.; Hahm, K. S. Biochim. Biophys. Acta 2003, 1645, 172.
(5) Porter, E. A.; Wang, X.; Lee, H. S.; Weisblum, B.; Gellman, S. H.
Nature 2000, 404, 565.
(6) Liu, D.; DeGrado, W. F. J. Am. Chem. Soc. 2001, 123, 7553.
(7) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm, M.
T.; Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 2794.
(37) Nijnik, A.; Madera, L.; Ma, S. H.; Waldbrook, M.; Elliott, M. R.;
Easton, D. M.; Mayer, M. L.; Mullaly, S. C.; Kindrachuk, J.; Jenssen,
H.; Hancock, R. E. W. J. Immunol. 2010, 184, 2539.
(8) Mowery, B. P.; Lee, S. E.; Kissounko, D. A.; Epand, R. F.; Epand,
R. M.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. J. Am. Chem. Soc.
2007, 129, 15474.
(9) Tew, G. N.; Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll,
P. J.; Klein, M. L.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 5110.
(10) Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.;
Nusslein, K.; Tew, G. N. J. Am. Chem. Soc. 2008, 130, 9836.
(11) Kuroda, K.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127, 4128.
(12) Arnt, L.; Nusslein, K.; Tew, G. N. Polym. Sci., Part A: Polym.
Chem. 2004, 42, 3860.
(13) Ilker, M. F.; Nusslein, K.; Tew, G. N.; Coughlin, E. B. J. Am.
Chem. Soc. 2004, 126, 15870.
(14) Gabriel, G. J.; Madkour, A. E.; Dabkowski, J. M.; Nelson, C. F.;
Nusslein, K.; Tew, G. N. Biomacromolecules 2008, 9, 2980.
(15) Arnt, .; I, L.; Tew, G. N. J. Am. Chem. Soc. 2002, 124, 7664a.
D
dx.doi.org/10.1021/ja303304j | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX