Lipidated cyclic γ-Aapeptides display both antimicrobial and anti-inflammatory activity

Article abstract of DOI:10.1021/cb4006613

Antimicrobial peptides (AMPs) are host-defense agents capable of both bacterial membrane disruption and immunomodulation. However, the development of natural AMPs as potential therapeutics is hampered by their moderate activity and susceptibility to prote

Full text of DOI:10.1021/cb4006613

Articles
pubs.acs.org/acschemicalbiology
Lipidated Cyclic γ‑AApeptides Display Both Antimicrobial and Anti-
inflammatory Activity
Yaqiong Li,†,⊥ Christina Smith,‡,⊥ Haifan Wu,† Shruti Padhee,† Namitha Manoj,‡ Joseph Cardiello,‡
Qiao Qiao,† Chuanhai Cao,§ Hang Yin,*,‡ and Jianfeng Cai*,†
†Department of Chemistry, University of South Florida, 4202 E. Fowler Avenue, Tampa, Florida 33620, United States
‡Department of Chemistry & Biochemistry and the BioFrontiers Institute, University of Colorado at Boulder, Boulder, Colorado
80309-0596, United States
§College of Pharmacy, University of South Florida, 4202 E. Fowler Avenue, Tampa, Florida 33620, United States
S
* Supporting Information
ABSTRACT: Antimicrobial peptides (AMPs) are host-defense agents
capable of both bacterial membrane disruption and immunomodulation.
However, the development of natural AMPs as potential therapeutics is
hampered by their moderate activity and susceptibility to protease
degradation. Herein we report lipidated cyclic γ-AApeptides that have
potent antibacterial activity against clinically relevant Gram-positive and
Gram-negative bacteria, many of which are resistant to conventional
antibiotics. We show that lipidated cyclic γ-AApeptides mimic the
bactericidal mechanism of AMPs by disrupting bacterial membranes.
Interestingly, they also harness the immune response and inhibit
lipopolysaccharide (LPS) activated Toll-like receptor 4 (TLR4)
signaling, suggesting that lipidated cyclic γ-AApeptides have dual roles as novel antimicrobial and anti-inflammatory agents.
Therefore, the dual functional roles of AMPs have potential
as new generation of anti-infective therapeutics.
apidly emerging resistance of antibiotics is a major public
1
Rhealth concern.
As such, natural antibiotics including
Nonetheless, AMPs are susceptible to proteolytic cleavage,8
hampering their development into novel therapeutics.
Peptidomimetics as an alternative approach may circumvent
these problems. This is because peptidomimetics, with modified
peptide backbones, are more stable against protease degrada-
tion. Examples of successful antimicrobial peptidomimetics
include β-peptides,9−11 peptoids,12,13 and others.4,8,14−24
Similar to AMPs, these peptidomimetics are designed to form
globally amphipathic structures upon interaction with lipid
bilayers, disrupting bacterial membranes. Although many
peptidomimetics have broad-spectrum antibacterial activity,
the reports of immunomodulatory responses are very rare.8,24
Tew et al. identified that AMP mimics antagonize lipoteichoic
acid (LTA)-activated Toll-like receptor 2 (TLR2) signaling.24
Here we present a new class of immunomodulatory
antimicrobial peptidomimetics that inhibit TLR signaling
without ligand interaction.
antimicrobial peptides have captured researchers’ attention.
Antimicrobial peptides (AMPs) are short cationic amphiphilic
peptides present in almost every living organism.2 Because of
their broad-spectrum antimicrobial activity, AMPs are a
promising new class of drug candidates.2 AMPs are effective
due to their ability to form a globally amphipathic structure
with segregated hydrophobic and cationic regions, capable of
disrupting bacterial membranes.2,3 Such disruption is rooted in
the physical properties of bacterial membranes, making the
development of resistance difficult.4 Additionally, AMPs are
known as host defense peptides (HDPs) for their role in
modulating host innate immunity and diminishing septic
responses after bacterial infection.2,5 The immunomodulatory
activities of AMPs are speculated to be a crucial contributor to
host defense, since the direct antimicrobial activity of AMPs is
often weak under physiological conditions.2,6,7 One of the most
important immune responses against bacterial infection is
lipopolysaccharide (LPS)-activated Toll-like receptor 4 (TLR4)
signaling. LPS is a characteristic component of Gram-negative
bacterial cell walls that activates the immune response through
TLR4. As such, AMPs may have dual roles as both antibiotic
and anti-inflammation agents in the treatment of pathogen
invasions. Because host inflammatory responses to bacterial
infection can lead to deadly septic shock, the current treatment
for severe infections involves the administration of both
antibacterial and anti-inflammatory drugs simultaneously.
We recently developed a new class of peptidomimetics
termed “γ-AApeptides” based on a chiral peptide nucleic
(PNA) backbone.25,26 Because of the versatility of γ-
AApeptides, we were able to synthesize potent antimicrobials
with broad-spectrum activities against both Gram-positive and
Received: September 1, 2013
Accepted: October 21, 2013
Published: October 21, 2013
© 2013 American Chemical Society
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Figure 1. The structures of lipidated cyclic γ-AApeptides. Two γ-AApeptides, HW-C-22-2 and HW-C-22-4, which are either linear or cyclic and do
not contain lipid tails, were used for comparison. Compounds are named according to their notebook codes.
Gram-negative bacteria.19,22,23,26 The design of antimicrobial γ-
AApeptides is straightforward, which is achieved through
joining of amphipathic γ-AApeptide building blocks. Herein,
we report the design and synthesis of a new generation:
lipidated cyclic γ-AApeptides. We show that this class of
compounds has potent and broad-spectrum antimicrobial
activity against both Gram-positive and Gram-negative bacteria,
including community-acquired multidrug-resistant pathogens.
Interestingly, several lipidated cyclic γ-AApeptides inhibit Toll-
like receptor 4 (TLR4) signaling and block production of the
proinflammatory cytokine tumor necrosis factor-α (TNF-α),
implying the potential for a new generation of antibiotic agents
with dual functionality.
RESULTS AND DISCUSSION
■
We have shown previously that antimicrobial AApeptides with
potent and broad-spectrum activity can be designed and
prepared easily by joining amphipathic AApeptide building
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a
Table 1. The Antimicrobial and Hemolytic Activities of Lipidated Cyclic γ-AApeptides
MIC, μg/mL (μM)
Gram-negative
Gram-positive
Oligomers
E. coli
K. pneumoniae
P. aeruginosa
S. epidermidis
E. faecalis
S. aureus
Hemolysis (HC50), μg/mL (μM)
HW-B-73
HW-B-77
HW-B-78
YL-1
>25 (16)
>25 (13)
>25 (11)
>25 (16)
>25 (13)
5 (3)
20 (12)
20 (10)
20 (9)
8 (5)
>25 (16)
>25 (13)
>25 (11)
20 (12)
>25 (13)
10 (6)
20 (12)
>25 (13)
>25 (11)
2 (1)
>25 (16)
>25 (13)
>25 (11)
5 (2)
20 (12)
>25 (13)
>25 (11)
5 (2)
250 (160)
250 (130)
180 (114)
150 (82)
200 (129)
100 (65)
100 (50)
120 (45)
YL-4
>25 (13)
5 (3)
>25 (13)
5 (3)
>25 (13)
5 (3)
>25 (13)
10 (6)
5 (3)
YL-12
YL-29
>25 (14)
10 (6)
10 (5)
3 (2)
25 (14)
10 (6)
5 (3)
5 (3)
YL-34
1 (0.6)
1 (0.6)
2 (1)
2 (1)
2 (1)
YL-36
2 (1)
3 (2)
5 (3)
3 (2)
2 (1)
HW-B-13
pexiganan
20 (10)
8 (3)
8 (4)
8 (4)
5 (3)
1 (0.5)
16 (6)
8 (3)
16 (6)
8 (3)
32 (12)
a
The microbial organisms used are Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13383), Pseudomonas aeruginosa (ATCC 27853),
methicillin-resistant Staphylococcus epidermidis (RP62A), vancomycin-resistant Enterococcus faecalis (ATCC 700802), and methicillin-resistant
Staphylococcus aureus (ATCC 33591). The minimum inhibitory concentration (MIC) is the lowest concentration that completely inhibits growth
after 24 h. HC50 is the concentration causing 50% hemolysis. Pexiganan8,29 and previously reported cyclic γ-AApeptide HW-B-1322 are included for
comparison. YL-36, the compound with the most potent and broad-spectrum activity, is highlight in italic type.
blocks together.19,22,23 This is because AApeptides formed by
this approach are capable of adopting globally amphipathic
structures, which are crucial for the disruption of bacterial
membranes.19,22,23 Moreover, cyclization22 or lipidation23 can
increase their potency. Cyclization reduces structure motility
and facilitates bacterial membrane disruption;22 while lipidation
encourages antimicrobial agent interactions with membranes.23
We therefore speculated that γ-AApeptides that are both
cyclized and lipidated would be more potent in killing bacterial
pathogens. In fact, lipidated cyclic peptides polymyxin27 and
daptomycin28 have been used as “last-resort” antibiotics to treat
infections caused by Gram-negative and Gram-positive bacteria,
respectively. However, the exploration of antimicrobial
peptidomimetics with lipidated cyclic structural motifs is scarce.
To test our hypothesis, a series of lipidated cyclic γ-AApeptides
(Figure 1) containing different numbers of amphiphilic γ-
AApeptide building blocks were designed and synthesized in
the solid phase. Their antibacterial activity was then measured
against a range of multidrug-resistant Gram-negative and Gram-
positive bacteria (Table 1).
We initially designed and synthesized lipidated cyclic γ-
AApeptides (HW-B-73, -77 and -78, Figure 1) in which the
lipid tail C16 is directly connected to the ring structure.
Unfortunately, these sequences only possess weak activity
against bacteria. We hypothesize that this is because the lipid
tail on the ring structure has limited orientation and therefore
cannot position itself for membrane insertion even after the
amphipathic ring contacts the bacterial membranes. Indeed, a
trend was apparent in other membrane-active lipidated cyclic
peptide antibiotics, including daptomycin and polymyxin B,
which contain at least two amino acid residues connecting both
lipid tails and ring structures.27,28 To test our hypothesis, we
moved the lipid tail outside the cyclic ring, leading to the
sequence YL-1. As expected, YL-1 showed strong activity
against Gram-positive bacteria, although it only contains three
amphiphilic building blocks. To determine how ring size affects
antibacterial activity, we then included more amphiphilic
building blocks into the ring structure. However, an additional
amphiphilic building block (YL-4) did not boost activity. A
similar phenomenon has been observed in cyclic peptoids12 and
other cyclic peptide antimicrobial agents,30,31 which normally
contain 6−8 residues in their ring structures. In fact, YL-4 has
weaker antimicrobial activity than YL-1, which may due to the
increased flexibility of the larger ring. This trend is also seen in
HW-B-73, HW-B-77, and HW-B-78.
We have previously observed that the exact amphiphilic
structures can affect the antimicrobial activity of a sequence.20
The reversal of cationic and hydrophobic groups results in
antimicrobial agents with different potency.20 It appears that
while the overall amphipathicity dominates antimicrobial
activity, the precise distribution of functional groups in
molecules will affect the strength of peptidomimetic interaction
with bacterial membranes. As such, YL-12, containing one
reversed amphiphilic building block in relation to YL-1, was
prepared. This sequence has improved antimicrobial activity
against both Gram-negative and Gram-positive bacteria. The
reversal of additional building blocks leads to the most potent
lipidated cyclic γ-AApeptides YL-34 and YL-36. Particularly,
YL-36, with a small amphipathic ring and a C16 lipid tail, shows
very potent activity against all tested drug-resist Gram-positive
and Gram-negative strains. It not only has much improved
antimicrobial activity over the AMP pexiganan but is also
superior when compared with the previously reported cyclic γ-
AApeptide HW-B-13 of much larger ring size, especially against
Gram-negative pathogens.
We hypothesize that lipidated cyclic γ-AApeptides are able to
kill bacteria through membrane disruption analogous to AMPs,
because they possess globally cationic amphipathic structures
and broad-spectrum activity against both Gram-positive and
Gram-negative bacteria. As such, a fluorescence microscopy
experiment was carried out to study the ability of the most
potent lead, YL-36, to affect the membranes of S. aureus (Gram-
positive) and E. coli (Gram-negative). Briefly, both bacteria
were stained with the membrane permeable dye 4′,6-diamidino-
2-phenylindole (DAPI) and the nonpermeable dye propidium
iodide (PI) in the absence or presence of YL-36 (Figure 2). YL-
36 treatment resulted in PI becoming visible using fluorescence
microscopy, suggesting bacterial membranes of both S. aureus
and E. coli were damaged. Aggregation of S. aureus after
treatment with YL-36 is observed, which is generally believed to
arise from the loss of membrane potential after the disruption
of membranes.19−23,26
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Figure 2. Fluorescence micrographs of S. aureus (A) and E. coli (B)
that are untreated or treated with 5 μg/mL lipidated cyclic γ-
AApeptide YL-36 for 2 h: (a1) control, no treatment, DAPI stained;
(a2) control, no treatment, PI stained; (b1) YL-36 treatment, DAPI
stained; (b2) YL-36 treatment, PI stained.
Figure 3. (a) Nitric oxide production in the presence of the lipidated
cyclic γ-AApeptides YL-1, YL-12, YL-29, and YL-36. Two γ-
AApeptides lacking alkyl tails, either linear (HW-C-22-2) or cyclic
(HW-C-22-4), were included to demonstrate the importance of γ-
AApeptide lipidation on their TLR activities. RAW 264.7 cells were
treated with 20 ng/mL LPS and varying concentrations of lipidated
cyclic γ-AApeptides in a 96-well plate. Data is normalized to 20 ng/mL
LPS as 100% activation, and untreated cells as 0% activation. (b) Dose-
dependent inhibition of nitric oxide production by YL-36. This
experiment was performed as described in part a, and a dose response
curve was obtained for calculation of an IC50 value.
In addition to bacterial membrane disruption, some AMPs
are speculated to modulate the immune system through TLR
signaling.32 Since lipidated cyclic γ-AApeptides were designed
to mimic AMPs, we anticipated that they may be also capable of
harnessing immune responses. As such, YL-1, YL-12, YL-29,
YL-34, and YL-36 were also assessed for the ability to modulate
the LPS-induced TLR4 signaling response by measuring nitric
oxide production (Figure 3a).33 Nitric oxide is produced as a
downstream inflammatory factor in all TLR signaling and is
part of the global inflammatory response. Figure 3a shows that
lipidated cyclic γ-AApeptides are a potent class of anti-TLR4
signaling agents, capable of reducing nitric oxide production.
To further investigate the anti-inflammatory capabilities of
lipidated cyclic γ-AApeptides, we chose the most potent
antimicrobial agent YL-36 for further investigation. The IC50
of YL-36 was obtained by treating RAW 264.7 cells with
varying concentrations of YL-36 in concert with 20 ng/mL
LPS. The resulting inflammatory response was monitored
through measurement of nitric oxide production.
YL-36 shows an inhibitory effect of the nitric oxide signaling
with an IC50 value of 1.98 μM (Figure 3b). HW-C-22-2 and
HW-C-22-4 demonstrate that lipidation increases the effective-
ness of γ-AApeptides as TLR signaling antagonists, because
neither construct is active under the tested concentrations
(Figure 3a,b). Nitric oxide is produced by TLR activation and
correlates to the level of inflammatory response. Additionally,
YL-36 shows little cytotoxicity up to 100 μM (Figure S3,
Supporting Information), suggesting that the inhibition of nitric
oxide is not due to the toxicity of YL-36. As such, we further
explored the ability of YL-36 to inhibit downstream NF-κB
activation and the production of pro-inflammatory cytokines.
Inhibition of the NF-κB activation (Figure 4a) was determined
by a previously developed secreted embryonic alkaline
phosphatase (SEAP) assay.34 Furthermore, the pro-inflamma-
tory cytokine tumor necrosis factor-α (TNF-α) is also strongly
inhibited (IC50 = 2.05 μM) (Figure 4b).35 Both NF-κB and
TNF-α are produced due to TLR activation. Their inhibition by
YL-36 shows that TLR signaling is being inhibited in a dose-
dependent fashion. Neither HW-C-22-2 nor HW-C-22-4 shows
inhibitory activities, further demonstrating that anti-inflamma-
tory activity depends on the lipidation of γ-AApeptides.
Tew et al. has previously elegantly identified antimicrobial
agents that reduce TLR-induced inflammatory response.8,24
Interestingly, our lipidated cyclic γ-AApeptides appear to have a
different mechanism of inhibition, not interacting with the TLR
ligands. If γ-AApeptides interacted with LPS, pretreatment with
YL-36 prior to LPS addition would abolish its anti-TLR4
activities. Our data though demonstrates that both pretreat-
ment and cotreatment with γ-AApeptides and LPS results in
complete antagonism of nitric oxide production (Figure 5),
indicating that γ-AApeptides do not bind to LPS directly.
Conclusion. As a new class of antimicrobial peptidomi-
metics that mimic AMPs, lipidated cyclic γ-AApeptides exhibit
potent and broad-spectrum activity against a range of
multidrug-resistant Gram-negative and Gram-positive bacteria.
Maybe even more importantly, lipidated cyclic γ-AApeptides
also imitate AMPs in their immunomodulatory capabilities.
These γ-AApeptides have been shown to antagonize the LPS
activated NF-κB signaling response and to potently suppress
release of harmful pro-inflammatory cytokines including tumor
necrosis factor-α (TNF-α). Additionally, lipidated cyclic γ-
AApeptides do not bind to TLR ligands, suggesting a novel
mechanism for immunomodulation. These peptidomimetics
provide an exciting new approach to treat bacterial infections by
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agents through harnessing immune responses. Furthermore,
because deregulation of TNF-α is also related to diseases such
as cancer and lupus erythematosus,36,37 lipidated cyclic γ-
AApeptides may also provide other therapeutic applications in
the future.
METHODS
■
General Experimental Methods. Rink amide MBHA resins
(200−400 mesh, 0.7 mmol/g) were purchased from Chem-Impex Int’l
Inc. Other chemicals were ordered from either Sigma-Aldrich or Fisher
1
Scientific and used without further purification. H NMR spectra of
the building blocks were obtained on an Agilent DD800 instrument.
The solid phase syntheses of the lipidated cyclic γ-AApeptides were
carried out in a peptide reaction vessel on a Burrell Wrist-Action
shaker, and the compounds were analyzed and purified using an
analytical and preparative Waters HPLC system, respectively. The final
products were dried in a Labcono lyophilizer. Molar masses were
identified using a Bruker AutoFlex MALDI-TOF mass spectrometer.
Solid Phase Synthesis of Lipidated Cyclic γ-AApeptides. The
syntheses of lipidated cyclic γ-AApeptides were carried out in the solid
phase as reported previously.19,22 For each coupling cycle, 20%
piperidine in DMF was used to remove the Fmoc protecting group,
followed by the coupling of 1.5 equiv of building blocks with 4 equiv of
HOBT (1-hydroxybenzotriazole monohydrate)/DIC (diisopropylcar-
bodiimide) in DMF for 6 h. The allyl group was removed by 0.2 equiv
of Pd(PPh3)4 in the presence of 10 equiv of PhSiH3/CH2Cl2 (2 h for
each, repeated twice). The exposed carboxyl group reacted with the N-
terminus of the sequence to complete the cyclization using PyBOP/
HOBT/DIPEA/DMF. The lipidated γ-peptides were cleaved from the
solid support in 50:48:2 TFA/CH2Cl2/TIS (triisopropylsilane) for 2 h.
The solvent was evaporated, and the sequences were analyzed and
purified using a Waters HPLC system monitored at 215 nm. The
desired fractions were collected and lyophilized, and their molecular
weights were confirmed by the Bruker AutoFlex MALDI-TOF mass
spectrometer.
Figure 4. (a) NF-κB activation in HEK 293 cells in the presence of
various γ-AApeptides. HEK 293 cells were stably transfected with
TLR4 and its accessory proteins, as well as a secreted embryonic
alkaline phosphatase (SEAP) reporter gene. Cells were plated in a 96-
well plate with 40 000 cells/well and treated with 20 ng/mL LPS and
YL-36. Data is normalized to 20 ng/mL LPS as 100% activation and
untreated cells as 0% activation. Increasing concentrations of YL-36
decrease transcription of SEAP in a dose-dependent fashion. HW-C-
22-2 and HW-C-22-4 were included for comparison. (b) YL-36
reduces production of TNF-α. TNF-α production was measured with
a monoclonal mouse ELISA. Data is normalized to 20 ng/mL LPS as
100% activation and untreated cells as 0% activation. Increasing
concentrations of YL-36 result in decreased production of TNF-α.
HW-C-22-2 and HW-C-22-4 were included for comparison.
Antimicrobial Assays.19,22 The lipidated cyclic γ-AApeptides
were tested for their antimicrobial activity against various microbial
organisms including E. coli (ATCC 25922), K. pneumonia (ATCC
13383), multidrug-resistant P. aeruginosa (ATCC 27853), methicillin-
resistant S. epidermidis (MRSE, RP62A), vancomycin-resistant E.
faecalis (ATCC 700802), methicillin-resistant S. aureus (ATCC
33592). The highest concentration of the tested AA-peptides was 25
μg/mL. The bacteria in 5 mL of medium were grown at 37 °C
overnight and then diluted to make a suspension of approximate 1 ×
106 CFU/mL. Aliquots of 50 μL of bacterial suspension were mixed
with 50 μL of medium containing different concentrations of lipidated
cyclic γ-AA-peptides. The plate was incubated at 37 °C overnight with
cell growth monitored by a Biotek Synergy HT microtiter plate reader
under the 600 nm wavelength. MIC was determined when the lowest
concentration of the compounds inhibit the cell growth completely in
24 h. The results were repeated at least three times with duplicates for
each time.
Hemolysis Assay.19,22 Freshly drawn, K2 EDTA treated human
red blood cells (hRBCs) were washed with PBS buffer twice and
centrifuged at 1000g for 10 min. After the clear supernatant was
removed, the cell pellets were mixed with serially diluted lipocyclic γ-
AApeptides in a 96-well plate. The plate was incubated at 37 °C for 1 h
and centrifuged at 3500 rpm for 10 min. The supernatant was
separated and diluted in PBS, and the absorbance was detected at 360
nm using a Biotek Synergy TH plate reader. Percent hemolysis =
(Abssample − AbsPBS)/(AbsTriton − AbsPBS) × 100%; 0% hemolysis
(negative control) was determined by mixing blood with PBS, and
100% hemolysis (positive control) was determined by mixing blood
with Triton X-100 (final concentration 0.1%). The results were
repeated at least three times with duplicates for each time.
Figure 5. YL-36 inhibits TLR4 activation independent of LPS binding.
RAW 264.7 cells were incubated for 30 min with 10 μM YL-36. After
incubation, pretreatment cells were washed with medium and activated
with 20 ng/mL LPS. Co-treatment cells were not washed and were
treated with 20 ng/mL LPS while YL-36 remained in the medium.
Antagonism for both cell treatments is 100%, suggesting that YL-36
does not bind to the ligand as a mode of inhibition. Data is normalized
to 20 ng/mL LPS as 100% activation and untreated cells as 0%
activation.
Fluorescence Microscopy.19,22 4′,6-Diamidino-2-phenylindole
dihydrochloride (DAPI, Sigma, >98%) and propidium iodide (PI,
Sigma) were used to stain the bacteria cells of E. coli or S. aureus. DAPI
is a DNA binding dye staining all bacterial cells regardless of their
exerting dual-functional roles: they are a new generation of
antibiotic agents that kill multidrug-resistant Gram-positive and
Gram-negative bacteria directly, as well as anti-inflammatory
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viabilities, and PI is an ethidium derivative that only can pass through
damaged bacterial membranes and intercalates with their nucleic acids.
Briefly, bacteria in mid-logarithmic phase were incubated with
lipidated cyclic γ-AA peptides (2 × MIC) for 2 h and then were
centrifuged at 3000g for 15 min. The bacteria cell pellets were
separated and then incubated with PI, followed by washing and
incubation with DAPI (each dye incubation was performed at 0 °C for
15 min in the dark). Controls were bacteria culture without peptides
following the same procedure described above. The stained bacteria
cells were observed under Zeiss Axio Imager Zloptical microscope
using the 100× oil-immersion objective.
Biosciences, CA, USA). The plate was read on a Beckman Coulter
DTX880 plate reader (Beckman Coulter, CA, USA). Data was
collected with absorbance at 450 nm. Data was normalized with the
ligand only control as 100% activation and the untreated cells as 0%
activation. Fold inhibition = [(Sample450nm − Untreated cells450nm)/
(Ligand Control450nm − Untreated cells450nm)]. The EC50 values were
calculated graphically using OriginPro v8.6 software.
Crystal Violet Toxicity Assay.34,35 Cells that were treated with
compound for nitric oxide experimentation were also tested for
compound toxicity using crystal violet stain. Cells were fixed for 20
min in 4% paraformaldehyde after the media was removed. After fixing,
formaldehyde was removed, and cells were incubated for 1 h with
0.05% crystal violet stain. After incubation, cells were rinsed with
deionized water and reconstituted in 100% methanol for 10 min. The
plate was read on a Beckman Coulter DTX880 plate reader (Beckman
Coulter, CA, USA). Data was collected with absorbance at 535 nm.
Data was normalized with the untreated cells control as 100% survival
Fluorescent Detection of Nitric Oxide.34,35 RAW 264.7 (mouse
leukemic monocyte macrophage cell line) cells were grown in RPMI
1640 medium containing 1% L-glutamine, 1% penicillin/streptomycin,
and 10% fetal bovine serum (FBS). Cells were plated in a 96-well plate
at 75 000 cells/well in complete RPMI 1640 medium and allowed to
grow overnight at 37 °C and 5% CO2 in a humidified incubator. The
media was removed, and cells were placed in unsupplemented RPMI
1640 medium. LPS (20 ng/mL) and the appropriate concentration of
lipidated cyclic γ-AApeptides (20 mM stock solutions in PBS) were
added to a final volume of 200 μL. PBS controls were included in each
experiment. Plates were incubated for 24 h, and then 100 μL of media
was transferred to a flat black 96-well microfluor plate (Thermo
Scientific, MA, USA). Following that, 10 μL of 0.05 mg mL−1 2,3-
diaminonaphthalene in 0.62 M HCl was added to the media and
incubated for 20 min in the dark. The reaction was quenched with 5
μL of 3.0 M NaOH, and the plate was read on a Beckman Coulter
DTX880 plate reader (Beckman Coulter, CA, USA). Data was
collected with excitation at 360 nm and emission at 430 nm. Data was
normalized with the ligand only control as 100% activation and the
and the blank wells as 0% survival. Fold inhibition = [(Sample535nm
Blank535nm)/(Untreated cells535nm − Blank535nm)].
−
ASSOCIATED CONTENT
* Supporting Information
■
S
Structures of lipidated cyclic γ-AApeptide building blocks,
synthesis and structures of lipidated cyclic γ-AApeptides,
toxicity of YL-36, MALDI analysis of lipidated cyclic γ-
AApeptides, experimental methods, HPLC traces, and NMR
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
untreated cells as 0% activation. Fold inhibition = [(Sample430nm
−
AUTHOR INFORMATION
Corresponding Authors
*E-mail: jianfengcai@usf.edu.
*E-mail: hubert.yin@colorado.edu.
■
Untreated cells430nm)/(Ligand Control430nm − Untreated cells430nm)].
The EC50 values were calculated graphically using OriginPro v8.6
software.
Secreted Embryonic Alkaline Phosphatase (SEAP) Reporter
of NF-κB Transcription.34,35 HEK293 (human embryonic kidney)
cells were grown in Dulbecco’s modified Eagle medium (DMEM) in
the presence of 1% penicillin/streptomycin, 10% fetal bovine serum
(FBS), and 1% L-glutamine. HEK293 cells are stably transfected with
human TLR4, as well as the required accessory proteins MD-2 and
CD14. Moreover, the cells also possess an optimized alkaline
phosphatase reporter gene under the control of a NF-κB inducible
promoter.38 Cells were first plated in a 96-well plate at 40 000 cells/
well in complete DMEM medium and allowed to grow overnight at 37
°C and 5% CO2 in a humidified incubator. Then the media was
removed, and cells were placed in Optimem + 0.5% FBS medium. LPS
(20 ng/mL) and the appropriate concentration of lipidated cyclic γ-
AApeptides were added to a final volume of 200 μL. PBS buffer was
included as control. Plates were then incubated for 24 h, and then
medium was assayed per the instructions of the Phospha-Light SEAP
reporter gene assay system (Applied Biosystems, NY, USA). The plate
was read on a Beckman Coulter DTX880 plate reader (Beckman
Coulter, CA, USA). Data was collected with luminescence at 430 nm.
Data was normalized with the ligand only control as 100% activation
and the untreated cells as 0% activation. Fold inhibition =
Author Contributions
⊥Y.L. and C.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the USF start-up fund (J.C.) and
National Institutes of Health (Grants GM101279 and
GM103843) (H.Y.).
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