Nobilamides A-H, long-acting transient receptor potential vanilloid-1 (TRPV1) antagonists from mollusk-associated bacteria

Article abstract of DOI:10.1021/jm101621u

New compounds nobilamides A-H and related known compounds A-3302-A and A-3302-B were isolated based upon their suppression of capsaicin-induced calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, nobilamide B

Full text of DOI:10.1021/jm101621u

ARTICLE
pubs.acs.org/jmc
Nobilamides AꢀH, Long-Acting Transient Receptor
Potential Vanilloid-1 (TRPV1) Antagonists from
Mollusk-Associated Bacteria
Zhenjian Lin,† Christopher A. Reilly,‡ Rowena Antemano,^ Ronald W. Hughen,§ Lenny Marett,†
||
Gisela P. Concepcion,^ Margo G. Haygood,# Baldomero M. Olivera,|| Alan Light,§ and Eric W. Schmidt*,†,
Departments of †Medicinal Chemistry, ‡Pharmacology and Toxicology, §Anesthesiology, and Biology, University of Utah,
Salt Lake City, Utah 84112, United States
^Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines
#Department of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University,
Beaverton, Oregon 97006, United States
S Supporting Information
b
ABSTRACT:
New compounds nobilamides AꢀH and related known compounds A-3302-A and A-3302-B were isolated based upon their
suppression of capsaicin-induced calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these
compounds, nobilamide B and A-3302-A, were shown to be long-acting antagonists of mouse and human TRPV1 channels,
abolishing activity for >1 h after removal of drug presumably via a covalent attachment. Other derivatives also inhibited the TRPV1
channel, albeit with low potency, affording a structureꢀactivity profile to support the proposed mechanism of action. While the
activities were modest, we propose a new mechanism of action and a new site of binding for these inhibitors that may spur
development of related analogues for treatment of pain.
’ INTRODUCTION
term.6 Vanilloids such as capsaicin bind at an intramembrane and
intracellular site located between TM segments 3 and 4, involving
Y511, S512, W549, and other residues.7 This site is distinct from
the channel pore loop segment, but binding of ligands to this site is
presumed to induce structural changes in the pore loop region such
that ions can pass through the tetrameric receptor pore. Antago-
nists that compete with vanilloids are among the major drug leads
so far identified. However, recently selective agonists that bind
directly to the channel region have also been identified.8 Because of
the varied physiological responses to individual agonists and
antagonists, there is still a need for further agents for clinical and
biological investigation.
More than 30 transient receptor potential (TRP) channels are
known, including many that are important in sensing stimuli such
as cold, heat, and pain. Among these, transient receptor potential
vanilloid-1 (TRPV1; VR1) is a nonselective cation channel that is a
major mediator of pain and inflammation.1 Stimuli such as heat,
protons, and chemical ligands provoke action potentials, leading
to the release of neurotransmitters and neuroactive peptides
[e.g., substance P, neurokinin A, and calcitonin gene-related
peptide (CGRP)] from peripheral and central nerve terminals.2
Many lines of experimental evidence indicate that selective TRPV1
antagonism could play a useful role in the treatment of chronic pain
and inflammatory hyperalgesia.3,4 Indeed, many such ligands have
been reported and have entered clinical and preclinical trials.4,5
Endovanilloids and the endogenously supplied ligand, capsaicin,
are potent TRPV1 activators that cause sensations of heat and pain
in the short term but lead to pain desensitization in the longer
TRPV1 is among the important channels found in dorsal root
ganglion (DRG) neurons, which transfer afferent signals such as
Received: December 23, 2010
Published: April 27, 2011
r
2011 American Chemical Society
3746
dx.doi.org/10.1021/jm101621u J. Med. Chem. 2011, 54, 3746–3755
|

Journal of Medicinal Chemistry
ARTICLE
pain, heat, and touch.9 These neurons also contain many other
types of channels and receptors, making them excellent models
for broad-net drug discovery assays. We employ an assay using a
primary culture of mouse DRG neurons containing many of the
different cell types normally present in the DRG of live mice.10
Cells are exposed to a series of treatments including KCl and
chemical extracts or pure compounds. By observing differences
in the resulting Ca2þ flux, we have discovered compounds that
act directly on channels and receptors important in transferring
information about pain, heat, touch, and other properties. For
example, using this assay, we recently reported the identification
of potent and selective ligands for the serotonin 5-HT2B
receptor.11
One of the advantages of the DRG assay is that cell types
containing distinct receptor and channel populations can be
pharmacologically distinguished. For instance, the application
of capsaicin differentiates nociceptors from other cell types. In
the course of screening bacterial extracts for DRG activity, the
organic extract of strain Streptomyces sp. CN48 produced a
novel effect; in addition to strongly increasing Ca2þ in DRG
neurons in response to KCl addition, the extract inactivated all
response to capsaicin, even 2 min after removal of the extract.
It was thus proposed that this extract might irreversibly
inactivate TRPV1 through a possibly novel mechanism. Here,
we report the discovery and structure elucidation of eight new
peptides, nobilamides AꢀH (4ꢀ11) and two related known
compounds A-3302-A (1) and A-3302-B (2), which produce
long-term (>1 h) inhibition of TRPV1. Their effects on
endogenously expressed and recombinant wild-type and mu-
tant human TRPV1 channels were assessed, showing that the
compounds function by a mechanism that appears to involve
covalent modification of TRPV1 through residues that con-
stitute an intracellular helix spanning TM4 and -5 and the pore
loop segment. Although the compounds are not exceptionally
potent, they provide lead structures for the development of a
new class of TRPV1 antagonists.
Structure Elucidation. By comparison of spectroscopic
data and physicochemical properties with literature reports,
two compounds 1 and 2 were identified as the previously
reported metabolites, A-3302-B (also known as TL-119) and
A-3302-A.12 Another isolated compound 3, N-acetyl-L-phe-
nylalanyl-L-leucinamide, has been synthesized but is not
previously known as a natural product;13 in this case, 3 is
instead composed of D-amino acids (see below).
The molecular formula of 4 was determined as C42H59N7O10
by high-resolution electrospray ionization mass spectrometry
1
(HRESIMS) and H and 13C NMR data (Tables 1 and 2),
indicating that it was larger than 1 by H2O. In addition, the
NMR data of 4 were largely identical to those of the known
compound 1. In fact, the only major difference was that the
threonine β-methine 1H and 13C resonances of 4 were shifted 0.8
and 5 ppm upfield in comparison to those of 1. This difference led
us to propose that 4 was a linear peptide lackingthe lactone linkage
found in 1. To confirm this hypothesis, we performed a complete
analysis of 1D and 2D NMR data (Supporting Information),
showingthat 4 contained the sameamino acidresidues in the same
order as 1: R,β-dehydrobutyrine (Dhb), alanine (Ala), valine
(Val), threonine (Thr), leucine (Leu), and two phenylalanines
(Phe). Additionaly, like 1, 4 was acetylated at its N terminus.
Compound 5 was assigned the molecular formula C43H61N7O10
on the basis of HRESIMS analysis and NMR experiments (Tables 1
and 2), making it larger than 2 by H2O and larger than 4 by CH2.
’ RESULTS AND DISCUSSION
Bioassay-Guided Purification. Strains CN48 and CT3a were
cultivated from dissected tissues of the mollusks Chicoreus nobilis
and Conus tribblei, respectively, from Cebu, Philippines. In-
formed local and national consent and intellectual property
agreements were obtained prior to performing this study. 16S
gene sequence analyses showed that both strains belong to the
genus Streptomyces. Sequences were deposited in GenBank
with accession numbers HQ696493 (CN48) and HQ696492
(CT3a).
Crude extracts of CN48 and CT3a were strongly active
in the DRG assay. The CN48 extract strongly activated
DRG cells upon addition of KCl, while that from CT3a was
strongly deactivating. Moreover, the CN48 extract blocked
activation by capsaicin. Despite these differences, HPLC analysis
showed that the two strains contained closely related families
of metabolites, which were further purified by bioassay- and
chemistry-guided fractionation. Following fermentation, cells
were pelleted by centrifugation, and the resulting broths were
subjected to HP20 resin adsorption chromatography. The
moderately polar fractions were further purified by C18 flash
chromatography followed by C18 HPLC to yield compounds
1ꢀ11.
1
Detailed analysis of 1Hꢀ H COSY and HMBC NMR experiments
indicated that 5 differed from 4 only by the absence of an acetyl
group; instead, a propionyl group (δH 1.98, 2H; 0.82, 3H) was
assigned to 5. Analysis of 2D NMR data of 5 confirmed the presence
of propionyl group and defined the amino acid sequence. Compound
5is identical to the known compound 2, except that it is linear instead
of a cyclic depsipeptide. Although 4 and 5 are only trivial derivatives
of the known compounds 1 and 2, careful HPLC analysis indicates
that they are indeed present in the fermentation media during the
normal course of bacterial growth in the same ratios found after
chemical isolation. Thus, they are naturally produced by these strains
under these experimental conditions and are not extraction artifacts.
Compound 6 was found to possess the molecular formula
C38H54N6O9 by analysis of HRESIMS and NMR data (Tables 1
and 2). The difference in the molecular formula of C4H5NO from
3747
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
3748
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
Table 2. 13C (125 MHz) NMR Data for Nobilamides AꢀH (4ꢀ11) in DMSO-d6
δC (mult.)
unit
Z-Dhb
no.
4
5
6
7
8
9
10a
11a
1
2
3
4
1
2
3
1
2
3
166.1 qC
129.4 qC
133.8 CH
15.8 CH3
171.5 qC
50.0 CH
19.9 CH3
171.2 qC
59.0 CH
32.6 CH
166.0 qC
128.5 qC
132.6 CH
14.2 CH3
171.4 qC
48.7 CH
18.3 CH3
171.0 qC
57.7 CH
31.0 CH
163.2 qC
126.0 qC
133.8 CH
15.3 CH3
170.1 qC
50.0 CH
17.9 CH3
171.3 qC
61.4 CH
28.6 CH
163.5 qC
126.3 qC
134.2 CH
15.5 CH3
170.1 qC
50.2 CH
17.9 CH3
171.3 qC
61.5 CH
29.2 CH
163.1 qC
126.2 qC
134.1 CH
15.2 CH3
168.7 qC
50.3 CH
17.8 CH3
170.2 qC
61.8 CH
28.9 CH
165.3 qC
128.0 qC
138.9 CH
15.7 CH3
173.1 qC
51.8 CH
18.6 CH3
174.8 qC
64.1 CH
31.3 CH
165.3 qC
127.9 qC
138.7 CH
15.7 CH3
173.2 qC
52.0 CH
19.1 CH3
174.5 qC
64.4 CH
31.1 CH
L-Ala
L-Val
174.6 qC
52.1 CH
17.8CH3
171.8 qC
57.7 CH
31.5 CH
4/5 21.3/19.8 CH3 19.5/18.3 CH3 20.1/18.9 CH3 19.5/19.4 CH3 19.8/19.5 CH3 19.6/19.9 CH3 20.8/20.4 CH3 20.7/20.3 CH3
D-a-Thr
1
2
3
4
1
2
3
4
170.4 qC
60.2 CH
68.8 CH
21.8 CH3
171.8 qC
55.9 CH
39.5 CH2
138.7 qC
170.2 qC
59.1 CH
67.7 CH
20.4 CH3
171.8 qC
54.0 CH
38.2 CH2
138.6 qC
128.4 CH
129.7 CH
126.7 CH
172.2 qC
51.9 CH
41.3 CH2
26.3 CH
170.3 qC
59.1 CH
67.9 CH
20.7 CH3
171.3 qC
54.7 CH
38.5 CH2
138.7 qC
128.7 CH
129.9 CH
126.8 CH
172.4 qC
48.3 CH
41.8 CH2
24.8 CH
168.1 qC
57.6 CH
73.9 CH
17.9 CH3
171.9 qC
55.0 CH
38.2 CH2
137.7 qC
128.0 CH
129.1 CH
126.5 CH
168.0 qC
58.3 CH
167.4 qC
58.1 CH
73.9 CH
17.5 CH3
171.4 qC
54.0 CH
37.3 CH2
135.1 qC
129.0 CH
129.8 CH
127.8 CH
171.4 qC
59.0 CH
171.3 qC
59.3 qC
73.4 CH
76.4 CH
76.3 CH
19.2 CH3
177.1 qC
64.4 CH
41.8 CH2
139.4 qC
130.4 CH
131.2 CH
128.9 CH
Propanone
64.6 CH
214.2 qC
26.2 CH3
17.6 CH3
17.8 CH3
172.2 qC
55.2 CH
17.7 CH3
177.1 qC
64.9 CH
L-Phe (L-Tyr)
37.0 CH2
130.6 qC
127.9 CH
115.3 CH
156.5 qC
172.6 qC
52.1 CH
42.0 CH2
139.8 qC
130.4 CH
131.2 CH
128.8 CH
Propanone
1 64.7 CH
2 215.3 qC
3 27.8 CH3
4 19.3 CH3
5,9 128.7 CH
6,8 129.9 CH
7
1
2
3
4
126.8 CH
172.4 qC
53.1 CH
42.7 CH2
25.8 CH
D-Leu
D-Phe
41.5 CH2
24.2 CH
5/6 23.8/24.7 CH3 22.8/22.6 CH3 23.5/22.6 CH3
19.5/23.5 CH3
171.6 qC
54.1 CH
1
2
3
4
172.0 qC
55.5 CH
39.0 CH2
138.6 CH
171.6 qC
54.7 CH
37.7 CH2
138.5 qC
128.0 CH
129.0 CH
126.7 CH
173.4 qC
28.6 CH3
10.3 CH3
172.2 qC
54.5 CH
172.0 qC
54.6 CH
37.6 CH2
137.4 qC
128.0 CH
129.0 CH
126.2 CH
169.9 qC
22.9 CH3
38.1 CH2
138.6 CH
128.6 CH
129.8 CH
126.8 CH
169.9 qC
23.3 CH3
37.6 CH2
138.2 qC
128.4 CH
130.1 CH
126.7 CH
169.7 qC
23.2 CH3
5,9 128.6 CH
6,8 129.8 CH
7
1
2
3
126.8 CH
169.9 qC
24.3 CH3
fatty acid
a Data were measured in CD3OD-d4.
4 was attributed to the absence of the Dhb residue. The 1H NMR
of 6 lacked the olefinic proton signal at about 6.5 ppm found in 4
and 5. Analysis of NMR data confirmed the absence of the Dhb
residue and allowed assignment of the amino acid sequence for 6.
Compound 7 was assigned the molecular formula C36H46N6O8
on the basis of HRESIMS analysis and NMR experiments (Tables 1
and 2). The NMR data of 7 were closely related to those of 1. The
chemical shifts of the Thr β-methine group were observed at δH 4.72
and δC 73.9, which indicated that 7 was a cyclic peptide with a
lactone linkage between the C-terminal amino acid and the hydroxyl
group of the Thr residue. Further analysis of the NMR data of 7
showed that, in comparison to 1, Leu was absent. A NOESY
correlation between protons at 4.48 and 8.42 ppm established the
connection between the two Phe residues in place of Leu.
The molecular formula of 8 was determined as
1
C42H57N7O10 by high-resolution ESIMS coupled with H
and 13C NMR data (Tables 1 and 2). The difference in the
molecular formula of an oxygen atom from 1 was attributed to
the presence of a tyrosine (Tyr) residue in place of Phe. The
1H NMR spectrum of 8 showed a pair of aromatic doublets δH
7.00, 6.62, which were assigned to Tyr. The 13C NMR
spectrum (Table 2) of 8 also indicated an oxygenated phenyl
carbon at δC 156.5. HMBC and NOESY NMR data were
consistent with the proposed structure of 8.
Compound 9 was assigned the molecular formula C25H35N5O6
on the basis of HRESIMS analysis and NMR experiments (Tables 1
and2).Thedifference in the molecular weight in comparison to 1was
attributed to the absence of two amino acid residues (Leu and Phe)
3749
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
Figure 2. Stability of TRPV1 inhibition. Compound 2 (375 μM) was
incubated with HEK-293 cells transfected with wild-type human
TRPV1. Response to capsaicin was measured over a 1 h timecourse
with washes at various intervals (x-axis). The change in fluorescent
response to Ca2þ (ΔF) was measured as a function of maximum
fluorescence or of fluorescence rate.
Figure 1. Time-dependent inhibition of TRPV1 by 5. HEK-293 cells
were transfected with human wild-type TRPV1 and pretreated with
compound 5 (375 μM), followed by application of capsaicin (25 μM).
For comparison, cells were cotreated with both agents. The change in
fluorescent response to Ca2þ (ΔF) was measured as a function of
maximum fluorescence or of fluorescence rate (n = 3).
Compounds 1ꢀ11 are closely related to each other, belonging
to a family that was previously only known from Bacillus
subtilis.15,16 There were a few noteworthy modifications. Com-
pound 6 is related to 4 just by the loss of Abu; this may be due to
enzymatic hydrolysis postsynthesis or to imperfect product
synthesis by biosynthetic enzymes. In comparison to 1, 7 is
missing a Leu that is within the peptide sequence itself. If the
compounds are indeed produced nonribosomally, this may be
due to module skipping.17 Compounds 10 and 11 are related to
the rest by loss of the two N-terminal amino acids, which are
instead replaced by an unprecedented ketone derivative. A
similar type of modification was recently described for an
unrelated natural compound.18
Capsaicin Antagonism in the Mouse DRG Assay. The
fluorometric calcium flux DRG assay allows for the simultaneous
study of 100ꢀ150 neurons with single application of com-
pounds. Multiple neuronal cell types are present in each well,
including a substantial fraction of nociceptors (∼30ꢀ50%); each
cell type has different combinations of receptors and channels
and exhibits a different pharmacological profile.10 In the standard
discovery assay, responses elicited by chemicals are normalized
by pulsing with 25 mM KCl, which leads to depolarization,
followed by washout, bringing cells back to baseline. Subse-
quently, samples of extracts or pure compounds are added to
observe any direct depolarizing effects, and then, samples are
added in tandem with 25 mM KCl to observe any increase or
decrease of depolarization. After a washout period, capsaicin
(a TRPV1 agonist) is added to differentiate nociceptors from
other cell types. Finally, a pulse of 100 mM KCl is added to
determine whether cells are still responding normally and to
obtain a value for maximum depolarization. By following changes
in intracellular Ca2þ over these steps, fine information about the
activity of extracts is revealed, enabling discovery of new agents.
Using the above procedure, application of a major fraction of
Streptomyces sp. CN48 extract led to a complete loss of response
to capsaicin, but subsequent addition of 100 mM KCl still
strongly depolarized all neurons in assay wells, indicating that
they otherwise were alive and functioning normally. In addition,
the extract was mildly stimulating to cells when coapplied with
25 mM KCl. In a pure compound test, 5 min after application of
purified 4 at a final concentration of 125 μM, the DRG cells were
depolarized by 25 mM KCl, and after that, a complete loss of
and an acetyl group from the N terminus of the peptide. Compatibly,
the NMR data (Tables 1 and 2) of 9 showed the absence of those
signals for the two amino acid residues and acetyl group. Analysis
of HMBC and NOESY NMR data confirmed the amino acid
sequence of 9.
Compounds 10 and 11 were isolated with very similar HPLC
retention times. The molecular formula for both was determined
as C29H41N5O7 by high-resolution ESIMS coupled with NMR
data (Tables 1 and 2). The 1H and 13C NMR data of 10 and 11
showed similar NMR signals (Tables 1 and 2), suggesting they
might be isomers of each other. In comparison, the 1H and 13
C
NMR data (Tables 1 and 2) to that of 9, two more methyl groups
(δH 2.14, H-3, δC 27.8, C-3 and δH 1.17, H-4, δC 19.3, C-4), and
a methine group (δH 3.42, H-1, δC 64.7, C-1) were present. The
distinctive methyl singlet at δH 2.14 corresponded to an R-keto
methyl group, but no ketone carbon was observed in the 13C
NMR spectrum (Table 2) of 10. In the HMBC spectrum of 10,
two strong correlations were observed from both H-3 and H-4 to
a ketone carbon at 215.3 ppm, indicating a ꢀCH(CH3)COCH3
fragment. Further HMBC correlations from H-1 to the R-C (δC
64.9) of Phe and from the R-H (δH 3.30) of Phe to C-1 indicated
the connection of these two partial structures. The same features
were found in the NMR data of 11. Therefore, compound 11 had
the same planar structure as 10.
Absolute configuration was assigned using Marfey's method.14
Compounds 4ꢀ11 were hydrolyzed, and the resulting amino
acids were converted to NR-(2,4-dinitro-5-fluorophenyl)-L-ala-
ninamide derivatives, which were characterized by HPLC in
comparison with authentic standards. Compounds 4ꢀ6 contain
L-Ala, L-Val, D-allo-Thr, D-Leu, and L- and D-Phe. In comparison
to 4ꢀ6, 7 is identical but lacks D-Leu; 8 contained L-Tyr in place
of L-Phe; and 9ꢀ11 lacked D-Phe and D-Leu. Compound 3
consisted solely of D-Leu-D-Phe. When both L- and D-Phe were
present, the order could not be ascertained from these experi-
ments. However, 1 was previously synthesized12 and was found
in both the cultures of CT3a and CN48. We propose that the
absolute configurations of 1ꢀ11 are identical. This hypothesis
was further supported by comparison with the configurations of
7ꢀ11, which were completely defined experimentally and in
which only one Phe residue was present. For 10 and 11, the
configurations of the terminal ketone moieties are speculative.
3750
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
response to capsaicin was observed. On the basis of these results,
we proposed that the nobilamides in the CN48 extract were
responsible both for increased depolarization of DRG cells and
for inhibition of response to capsaicin.
Nobilamides Induce Long-Term Changes in TRPV1. Two
likely mechanisms could explain the DRG results. In the first,
nobilamides could impact regulatory proteins that reduce cap-
saicin receptor activity. In the second, because of the long delay
prior to addition of capsaicin, it was possible that nobilimides
irreversibly inactivated capsaicin receptors. We therefore tested
individual compounds in assays using the human bronchial
epithelial cell line (BEAS-2B) cells, which stably overexpress
human TRPV1, primarily intracellularly.19ꢀ22 Direct competi-
tion experiments indicated that compounds 2 and 5 antagonize
the action of capsaicin on TRPV1 (Supporting Information),
with an apparent preference for cell surface-localized channels.
The onset of action was slow, and preincubation led to substan-
tially greater activity versus coapplication. However, with this cell
line, we could not determine whether this delayed effect might be
due to a slow on-rate (such as found in some irreversible
inhibitors) or whether cell penetration was a limiting factor for
these large peptides. Therefore, we used HEK-293 cells trans-
fected with human TRPV1. This cell line does not normally
express capsaicin-sensitive channels, and upon transfection,
TRPV1 is expressed largely on the cell surface, making it possible
to measure inhibition in the absence of potential confounding
effects. In this cell line, the potency of 5 was greatly increased
with preincubation of the compounds prior to capsaicin applica-
tion, in comparison to coapplication with capsaicin (Figure 1).
This observation was consistent with an irreversible binding
model. In a series of further experiments, the inhibitory activity of
5 was found to be stable through four washes taking place over a
60 min period (Figure 2). However, a slight rebound of activity
was noticed after 1 h in these human cells, indicating either a slow
reversibility or a replacement of inhibited TRPV1 by newly
synthesized protein.
To further examine this effect, we investigated the long-term
antagonism of mouse DRG neurons, in an assay adapted to allow
lengthy survival and monitoring of capsaicin responses in in-
dividual neurons (100ꢀ150 per well). In the first experiment,
mouse DRG neurons were incubated with compound 4, then
allowed to recover over a 3 h period. Although the capsaicin
response was initially abolished, recovery was observed after 3 h.
To determine whether recovery was due to new protein synth-
esis, TRPV1 recycling, or slow reversibility of binding, cells were
also treated with actinomycin D, cycloheximide, and brefeldin A
under several different conditions to inhibit transcription, trans-
lation, and trafficking of TRPV1 to the cell surface and subjected
to the same 3 h recovery experiment. Individual neurons were
followed for the entire duration of the experiment so that
recovery of single cells would be observable. Response to
capsaicin was restored after 3 h, indicating that channel synthesis
or turnover was likely not involved in recovery, and that instead
tightly or covalently bound 4 was slowly released over this time
course. Thus, it can be concluded that nobilamides are long-
acting antagonists of TRPV1, probably through a covalent
modification of the receptor. Because agonists that act for >15
min on TRPV1 have previously been termed “essentially
irreversible”,8 we propose that nobilamides represent a class of
essentially irreversible antagonists.
Figure 3. Mutational analysis of human TRPV1 inhibition. (A)
Compounds 2 (375 μM) and 5 (375 μM) were preincubated with
HEK-293 cells transfected with wild-type and mutant TRPV1 chan-
nels. Inhibition of the capsaicin response was compared. The change in
fluorescent response to Ca2þ (ΔF) was measured as a function of
maximum fluorescence or of fluorescence rate. (B) Side view of a
homology model of a single subunit of human TRPV1.25 Highlighted
residues involved in capsaicin binding (green) and nobilamide binding
(yellow, Cys; red, Phe) as well as residues whose mutation does not
affect nobilamide binding. (C) Side view of a homology model of the
TRPV1 tetramer pore loop region showing residues and intramole-
cular distance between residues involved in capsaicin or nobilamide
binding (colors as in B). The ion pore runs through the middle of this
diagram and is presumably blocked by nobilamide binding between
F660 and/or C578 and C621, either within or between subunits of the
functional tetramer.
Nobilamides Block TRPV1 Channel at a Novel Site. Because
the inhibitory nobilamides contain the modest electrophile,
3751
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
dehydrobutyrine, it seemed possible that the compounds might
covalently modify nucleophiles in TRPV1, leading to inhibition.
Indeed, we reduced the dehydrobutyrine residue of 1 with H2,
and the resulting 1a was completely inactive. Candidate nucleo-
philes included cysteine (Cys) residues in the pore loop region,
which were previously shown to be important redox-sensitive
residues. TRPV1 is very active in reducing conditions, and its
activity greatly decreases through Cys disulfide formation in the
channel under more oxidizing conditions.23 In addition, other
TRP channels have been found, which are agonized or antag-
onized by relatively nonspecific electrophiles.24
nobilamide concentrations (above 200 μM, in comparison to
an activating capsaicin concentration of 2ꢀ25 μM in these cell
lines). However, this activity was highly selective, and slight
differences in structure led to complete abrogation of activity. Of
these compounds, only 1, 2, 4, and 5 exhibited inhibitory activity.
In fact, very slight modifications led to large activity differences.
Compounds 1 and 4 exhibited relatively low activity, with IC50
values of 1320 and 1665 μM, respectively. However, 2 and 5 were
much more potent, with IC50 values of 227 and 275 μM,
respectively. The only difference between 2/5 and 1/4 is that
the former are longer by a single methylene group. By contrast,
the existence of a constraining ester does not appear to be highly
important in conferring activity. Neither 6 nor 1a exhibited
detectable activity, indicating the importance of the intact
dehydrobutyrine residue. Additionally, 7ꢀ11, which differed
only by length or residue in the side chain, were totally inactive.
Strikingly, the only difference between active 1 and inactive 8 was
the presence of OH (Tyr) in place of H (Phe) in the side chain.
Compound 7 was lacking only a single Leu residue in the side
chain. These results reinforce the selective nature of inactivation
and indicate that inhibition is not due to a simple and nonspecific
covalent modification.
In conclusion, these results demonstrate that nobilamides
antagonize TRPV1 and highlight the possibility of engineering
more potent analogues that block the TRPV1 channel in this
novel manner. Very slight differences in amino acids or in the N
terminus of the peptides exert large effects on activity, so that
synthesis of analogues is promising. Moreover, within the natural
derivatives, the amino acid sequence is relatively fixed; there is
much room for modification by making substitutions. The
presence of D-amino acids in key positions is also a great benefit
to design of more potent analogues, since D-peptides are known
to be more stable than their L-counterparts in human use.32
Finally, irreversibility itself may prove to be an advantage.
To test the site of binding and the possibility of covalent
modification, human embryonic kidney cell line (HEK-239) cells
overexpressing mutant human TRPV1 variants were treated with
compounds 2 and 5 (Figure 3). Six mutants were used. Of these,
three were inhibited by 2 and 5 in a manner similar to wild type,
while three exhibited significantly reduced inhibition by 2 or 5.
Two Cys mutants (C578A and C621A) and the F660A mutant,
adjacent to the ion conducting pore of TRPV1,25 led to a
complete loss of TRPV1 antagonism. These results suggest that
2 and 5 bind directly to the pore loop segment of the channel,
where they act as channel blockers by either modifying local
protein dynamics required for activation and/or ion flux or as a
“molecular blockade” via the formation of covalent adducts that
possibly bridge TRPV1 subunits to prevent ion flux from
occurring. It is curious that removing either Cys residue abolishes
the antagonist response, which defies simple models of single
alkylation. Moreover, incubation of nobilamides with glutathione
did not impact activity, indicating that the dehydrobutyrine
residue is at most a modestly active electrophile that requires
appropriate steric directing by adjacent residues such as F660 on
TRPV1. We propose that nobilamides covalently inactivate
TRPV1 based upon: (1) the lack of activity when the electrophile
Dhb was reduced or absent; (2) the requirement for Cys residues
in the binding site; (3) the slow onset time for activity; and (4)
the extremely long duration of binding. Whether binding is
covalent or not and indeed whether or not it involves 1:1
stoichiometry, the compounds essentially irreversibly inhibit
TRPV1 at a novel site of action.
Covalent Cys modification is important for rodent TRPV1
agonists such as allicin and nitric oxide, which act primarily on
C157 of the rat proteins.26,27 These activating effects are
relatively short-lived and readily reversible in comparison to
the inactivating effect of nobilamides, which are long lasting and
act in a wholly different region of the protein. By further contrast,
a pore loop Cys residue (C621) impacting nobilamide activity in
human TRPV1 potentiates the response of the rat channel to
heat.28 F660 is required for acid sensitivity, and mutations of this
residue completely abolish this response while maintaining
capsaicin sensitivity.29 Very recently, data were obtained indicat-
ing that F660 is probably primarily involved in gating the
response to protons.30 It is interesting that nobilamides appar-
ently directly act on or are affected by amino acids that potentiate
the response to important additional TRPV1 activators. On the
basis of these results, we propose that the nobilamides block the
pore such that they antagonize activation by many different
activators including capsaicin, heat, and protons. Direct channel
blocking antagonists, such as tetrabutylammonium,31 have been
previously described, but these in general exhibit lower potency
than nobilamides, lack selectivity, and are readily reversible.
StructureꢀActivity Relationships. Persistent human and
mouse TRPV1 antagonism was observed at relatively high
’ EXPERIMENTAL SECTION
General. UV spectra were obtained using a Perkin-Elmer Lambda2
UV/vis spectrometer. IR spectra were recorded on a JASCO FT/IR-420
spectrometer. NMR data were collected using either a Varian INOVA
500 (1H 500 MHz, 13C 125 MHz) NMR spectrometer with a 3 mm
Nalorac MDBG probe or a Varian INOVA 600 (1H 600 MHz, 13C 150
1
MHz) NMR spectrometer equipped with a 5 mm H[13C,15N] triple
resonance cold probe with a z-axis gradient and utilized residual solvent
signals for referencing. High-resolution mass spectra (HRMS) were
obtained using a Bruker (Billerica, MA) APEXII FTICR mass spectro-
meter equipped with an actively shielded 9.4 T superconducting magnet
(Magnex Scientific Ltd., United Kingdom), an external Bruker APOL-
LO ESI source, and a Synrad 50W CO2 CW laser. All compounds were
assessed to be >99% pure by HPLC with DAD and MS detectors.
Fermentation and Extraction. Strains CN48 and CT3a were
each individually grown at 30 ꢀC with shaking at 200 rpm in a 10 L
fermentor containing 10 L of ISP2 medium (0.2% yeast extract, 1% malt
extract, 0.2% glucose, and 2% NaCl). After 8 days, the broth was
centrifuged, and the supernatant was extracted with HP-20 resin for
4 h. The resin was filtered through cheesecloth, washed with water to
remove salts, and eluted with MeOH to yield the crude extract.
Purification. The crude extract (650 mg) of CN48 was separated
into five fractions (Fr1ꢀFr5) on a C18 column using gradient elution of
MeOH in H2O (50, 60, 70, 80, and 100%). Fr4 eluting in 80% MeOH
was further purified by C18 HPLC using 85% MeOH in H2O to obtain 4
(100.0 mg) and 6 (4.0 mg), and two further fractions, Fr4-3 and Fr4-4.
Fr4-3 was further purified by C18 HPLC using 37% CH3CN in H2O with
3752
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
0.1% TFA to obtain 5 (2.0 mg), 7 (1.0 mg), and 8 (1.2 mg). Fr4-4 was
further purified by C18 HPLC using 45% CH3CN in H2O with 0.1%
TFA to obtain 1 (150.0 mg). Fraction Fr5 was further purified by C18
HPLC using 55% CH3CN in H2O with 0.1% TFA to obtain 2 (1.0 mg).
The crude extract (350 mg) of CT3a was separated into five fractions
(Fr1ꢀFr5) on a C18 column using gradient elution of MeOH in H2O
(20, 40, 60, 70, and 80%). Fr4 eluting in 70% MeOH was further purified
by C18 HPLC using 39% CH3CN in H2O to obtain 9 (1.5 mg), 10 (2.0
mg), and 11 (3.0 mg). Compound 3 was obtained from fraction Fr2 by
C18 HPLC using 25% CH3CN in H2O. In addition, compounds 4 (1.0
mg) and 1 (0.4 mg) were also isolated from fraction Fr5 of CT3a.
Compound 1a. Compound 1 (1.0 mg) in methanol (2.0 mL) was
treated with a balloon of H2 gas and 10% palladium on carbon (3 mg)
overnight. The reaction mixture was filtered through silica gel, evapo-
rated to dryness, and purified by HPLC (80% methanol in H2O with
0.1% TFA) to give 1a (0.4 mg; 40% isolated yield); white solid. 1H NMR
(DMSO, 500 MHz), L-Phe: δ 4.63 (1H, m, R-H), 3.27 (2H, m, β-H),
6.96ꢀ7.27 (5H, m, Ph-H); D-Phe: δ 4.65 (1H, m, R-H), 2.68 (1H, dd,
J = 14.1, 5.0 Hz, β-H1), 6.96ꢀ7.27 (5H, m, Ph-H); 4.67 (1H, dd, J = 9.5,
5.2 Hz, R-H, Phe), 4.45 (1H, t, J = 7.6 Hz, R-H, Leu), 2.92 (1H, dd, J =
14.1, 10.0 Hz, β-H2), 6.96ꢀ7.27 (5H, m, Ph-H); L-Val: δ 3.94 (1H, m,
R-H), 2.06 (1H, m, β-H), 0.86 (3H, d, J = 6.5 Hz, γ-Me), 0.79 (3H, d, J =
6.5 Hz, γ-Me); But: δ 4.23 (1H, m, R-H), 1.57 (2H, m, β-H), 0.72 (3H,
t, J = 7.5 Hz, γ-Me); L-Ala: δ 3.76 (1H, m, R-H), 1.04 (3H, d, J = 6.8 Hz,
β-Me); D-Leu: δ 3.92 (1H, m, R-H), 1.22 (2H, m, β-H), 1.20 (1H, m, γ-
H), 0.81 (6 H, d, J = 6.5 Hz, δ-Me); D-a-Thr: δ 4.20 (1H, m, R-H), 4.21
(1H, m, β-H), 1.30 (3H, d, J = 6.5 Hz, γ-Me); δ 1.91 (3H, s, acetyl).
ESIMS m/z 806 [M þ H]þ, 828 [M þ Na]þ.
and 13C NMR, see Tables 1 and 2. HRESIMS m/z 842.4095 [M þ Na]þ
(calcd for C42H56N7O10Na, 842.4059, δ = 4.3 ppm).
20
Nobilamide F (9). White solid; [R]D ꢀ5 (c 0.1, DMSO). UV
(MeOH) λmax (log ε) 210 (3.8), 255 (1.4) nm. IR (film) νmax: 3124,
1703, 1672, 1688, 1452, 989 cmꢀ1. 1H and 13C NMR, see Tables 1 and 2.
HRESIMS m/z 502.2666 [M þ H]þ (calcd for C25H36N5O6, 502.2660,
δ = 1.2 ppm).
20
Nobilamide G (10). Colorless solid; [R]D ꢀ24 (c 0.1, DMSO).
UV (MeOH) λmax (log ε) 210 (4.0), 255 (1.3) nm. IR (film) νmax: 3278,
2926, 1754 1704, 1655, 1508, 1458, 983 cmꢀ1. 1H and 13C NMR, see
Tables 1 and 2. HRESIMS m/z 572.3081 [M þ H]þ (calcd for
C29H42N5O7, 572.3079, δ = 0.4 ppm).
20
Nobilamide H (11). Colorless solid; [R]D ꢀ27 (c 0.1, DMSO).
UV (MeOH) λmax (log ε) 210 (4.0), 255 (1.4) nm. IR (film) νmax: 3290,
2920, 1696, 1664, 1552, 1520, 1256, 1032 cmꢀ1. 1H and 13C NMR, see
Tables 1 and 2. HRESIMS m/z 572.3079 [M þ H]þ (calcd for
C29H41N5O7, 572.3079, δ = 0 ppm).
DRG Assay. DRG cells from cervical and lumbar regions were
obtained from C57B1 mice and used in an assay with bacterial culture
extracts and pure compounds as previously described.10 Briefly, DRG
cells were suspended in medium with additives and loaded with Fura-2 a.
m. (Molecular Probes), a fluorescent dye used to measure intracellular
calcium levels. Experiments were performed at room temperature
(20ꢀ25 ꢀC) in a 24-well plate format using fluorescence microscopy.
Individual cells were treated as single samples, so that the individual
responses of diverse neuron subtypes from the DRG could be examined.
After baseline measurements, the cells were treated with 25 mM KCl
solution and then washed. After return to baseline, bacterial extracts,
fractions, or pure compounds were applied. This solution was then later
replaced with 25 mM KCl solution 5 min later. To differentiate pain-
sensing and TRPV1-expressing neurons from other neuronal types,
capsaicin was applied after return to baseline of the extracts. Finally,
additional pulses of 25 mM KCl or 100 mM KCl were applied to
determine whether cells were still viable with normal action potentials.
In long-term TRPV1 antagonism test, the cells were treated with
capsaicin solution (100 or 200 nM) and then washed. After fluorescence
returned to baseline, test compounds (125 μM) were applied and
incubated for 5 min. The solution was decanted, and capsaicin solution
(100 or 200 nM) was added, followed by a wash. After 3 h, another
capsaicin solution was applied. Finally, an additional pulse of KCl
(100 mM) was applied to determine whether cells were still viable with
normal action potentials. To test for protein synthesis and trafficking,
cycloheximide (10 μg/mL), actinomycin D (6.3 μg/mL), and brefeldin
A (5.6 μg/mL) were added either before or after compound application.
TRPV1 Assays. Cell line-based assays were performed using a BMG
Labtech NOVOStar fluorescence plate reader equipped with a plate-to-
plate reagent delivery system. BEAS-2B cells that stably overexpress
human TRPV1 have been previously described.19ꢀ22 These cells express
TRPV1 primarily intracellularly on the endoplasmic reticulum and
exhibit an EC50 for capsaicin-induced calcium flux of 1ꢀ2 μM. Tran-
siently transfected human embryonic kidney (HEK-293) cells were used
for experiments involving overexpressed TRPV1 and mutants thereof. In
these cells, TRPV1 is expressed primarily on the cell membrane.
BEAS-2B cells were grown to confluence in fibronectin/collagen/
albumin coated 96-well plates in LHC-9 growth media, as previously
described.19ꢀ22 Cells were prepared for the calcium flux assay by
replacing the growth media with a 1:1 solution of LHC-9 and Fluo
4-Direct (Invitrogen) reagent containing Fluo 4 a.m., pluronic F-127,
probenecid, and a proprietary quencher dye. Cells were incubated at
room temperature (∼22 ꢀC) for 1 h in the dark and subsequently
washed by replacing the loading solution with LHC-9 containing 1 mM
water-soluble probenecid (Invitrogen) and 750 μM Trypan Red (ATT
Bioquest). For preincubation experiments, test compounds were then
added to the wash solutions in varying concentrations (described in
N-Acetyl-L-phenylalanyl-L-leucinamide (3). Colorless solid.
1H NMR (CDCl3, 500 MHz): δ 7.18ꢀ7.28 (5H, m, Ph-H, Phe), 4.67
(1H, dd, J = 9.5, 5.2 Hz, R-H, Phe), 4.45 (1H, t, J = 7.6 Hz, R-H, Leu),
3.17 (1H, dd, J = 14.1, 5.0 Hz, β-H1, Phe), 2.85 (1H, dd, J = 14.1, 10.0
Hz, β-H2, Phe), 1.88 (3H, s, acetyl group), 1.71 (1H, m, γ-H, Leu), 1.64
(2H, m, β-H, Leu), 0.96 (3H, d, J = 6.5 Hz, Me, Leu), 0.92 (3H, d, J = 6.5
Hz, Me, Leu). 13C NMR (CDCl3, 125 MHz): δ 174.5 (C, COOH, Leu),
172.6 (C, CdO, Phe), 171.9 (C, CdO, acetyl group), 137.3 (C, PhꢀC,
Phe), 129.1 (CH, PhꢀCH, Phe), 128.2 (CH, PhꢀCH, Phe), 126.5
(CH, PhꢀCH, Phe), 54.7 (CH, R-CH, Phe), 50.9 (CH, R-CH, Leu),
40.5 (CH2, β-CH2, Leu), 37.7 (CH2, β-CH2, Phe), 24.8 (CH, γ-CH,
Leu), 22.2 (CH3, acetyl group), 21.1 (CH3, Leu), 20.7 (CH3, Leu).
ESIMS m/z 321 [M þ H]þ.
20
Nobilamide A (4). White solid; [R]D ꢀ20 (c 0.1, DMSO). UV
(MeOH) λmax (log ε) 210 (3.9), 243 (1.2) nm. IR (film) νmax: 3272,
2978, 1808, 1712, 1696, 1648, 1568, 1553, 1537, 1112, 984 cmꢀ1. 1H
and 13C NMR, see Tables 1 and 2. HRESIMS m/z 844.4232 [M þ Na]þ
(calcd for C42H58N7O10Na, 844.4216, δ = ꢀ1.9 ppm).
20
Nobilamide B (5). White solid; [R]D þ3 (c 0.01, DMSO). UV
(MeOH) λmax (log ε) 210 (4.0), 243 nm (1.3). IR (film) νmax: 3276,
2923, 1753, 1692, 1630, 1568, 1553, 1538, 1107, 985 cmꢀ1. 1H and 13
C
NMR, see Tables 1 and 2. HRESIMS m/z 858.4418 [M þ Na]þ (calcd
for C43H60N7O10Na, 858.4372, δ = ꢀ5.4 ppm).
20
Nobilamide C (6). White solid; [R]D ꢀ15 (c 0.1, DMSO). UV
(MeOH) λmax (log ε) 210 (3.9), 243 (1.1) nm. IR (film) νmax: 3142,
2936, 1652, 1648, 1520, 1510, 1272, 1016 cmꢀ1. 1H and 13C NMR, see
Tables 1 and 2. HRESIMS m/z 761.3889 [M þ Na]þ (calcd for
C38H53N6O9Na, 761.3844, δ = 5.9 ppm).
20
Nobilamide D (7). White solid; [R]D ꢀ24 (c 0.1, DMSO). UV
(MeOH) λmax (log ε) 210 (3.8), 255 (1.5) nm. IR (film) νmax: 3281,
2930, 1750, 1703, 1641, 1563, 1537, 1521 1265, 984 cmꢀ1. 1H and 13
C
NMR see Tables 1 and 2. HRESIMS m/z 713.3334 [M þ Na]þ (calcd
for C36H45N6O8Na, 713.3270, δ = ꢀ9.0 ppm).
20
Nobilamide E (8). White solid; [R]D ꢀ40 (c 0.01, DMSO). UV
(MeOH) λmax (log ε) 210 (3.9), 256 (1.1), 276 (0.8) nm. IR (film) νmax
:
3296, 3062, 2968, 1664, 1648, 1563, 1547, 1531, 1249, 1016 cmꢀ1. 1H
3753
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
figure legends). After 30 min of incubation of cells at room temperature,
assays were initiated by addition of capsaicin to a final concentration of
2.5 μM at 37 ꢀC (or 25 μM for HEK-293 cells). If test compounds were
not preincubated, they were added concurrently with capsaicin. Changes
(4) Malmberg, A. B., Bley, K. R., Eds. Turning up the Heat on Pain:
TRPV1 Receptors in Pain and Inflammation; Progress in Inflammation
Research; Birkh€auser-Verlag: Boston, 2005.
(5) Gunthorpe, M. J.; Chizh, B. A. Clinical development of TRPV1
antagonists: Targeting a pivotal point in the pain pathway. Drug
Discovery Today 2009, 14, 56–67.
in intracellular fluorescence (resulting from changes in cytosolic Ca2þ
)
were monitored for 1 min. Data were quantified in two ways. Rates were
(ΔF/s) determined in comparison to the initial linear response observed
in a control consisting of capsaicin only treated cells. The magnitude of
the response (ΔFmax) observed for the entire 1 min time period was also
calculated in comparison to control.
Human TRPV1 was cloned into the pcDNA3.1D V5/His vector
(Invitrogen) and modified using the QuickChange site-directed muta-
genesis kit (Stratagene). Mutations were confirmed by DNA sequen-
cing. Plasmids (200 ng/well) were transfected into HEK-293 cells grown
to confluence in 1% gelatin-coated 96-well plates using Lipofectamine
2000 (2:1 lipid:DNA ratio) prepared in OptiMEM media. Cells were
cultured in the presence of the reagent for 4 h and cultured for 48 h in
DMEM:F12 þ 5% FBS. After this time, cells were processed and assayed
for calcium flux as described above, except that the fluorophore loading
steps were performed at 37 ꢀC.
(6) Jara-Oseguera, A.; Simon, S. A.; Rosenbaum, T. TRPV1: On the
road to pain relief. Curr. Mol. Pharmacol. 2008, 1, 255–269.
(7) Thomas, K.; Ethirajan, M.; Shahrokh, K.; Lee, J.; Cheatham, T.,
III; Sun, H.; Yost, G.; Reilly, C. Structure-activity relationship of
capsaicin analogs and transient receptor potential vanilloid 1-mediated
human lung epithelial cell toxicity. J. Pharmacol. Exp. Ther. 2011,
337, 400–410.
(8) Bohlen, C. J.; Priel, A.; Zhou, S.; King, D.; Siemens, J.; Julius, D. A
bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by
targeting the outer pore domain. Cell 2010, 141, 834–845.
(9) Gallego-Sandin, S.; Rodriguez-Garcia, A.; Alonso, M. T.; Garcia-
Sancho, J. The endoplasmic reticulum of dorsal root ganglion neurons
contains functional TRPV1 channels. J. Biol. Chem. 2009, 284, 32591–32601.
(10) Peraud, O.; Biggs, J. S.; Hughen, R. W.; Light, A. R.; Concep-
cion, G. P.; Olivera, B. M.; Schmidt, E. W. Microhabitats within
venomous cone snails contain diverse actinobacteria. Appl. Environ.
Microbiol. 2009, 75, 6820–6826.
’ ASSOCIATED CONTENT
(11) Lin, Z.; Antemano, R. R.; Hughen, R. W.; Tianero, M. D. B.;
Peraud, O.; Haygood, M. G.; Concepcion, G. P.; Olivera, B. M.; Light,
A.; Schmidt, E. W.; Pulicatins, A.-E. neuroactive thiazoline metabolites
from cone snail-associated bacteria. J. Nat. Prod. 2010, 73, 1922–1926.
(12) Kitajima, Y.; Waki, M.; Shoji, J.; Ueno, T.; Izumiya, N. Revised
structure of the peptide lactone antibiotic, TL-119 and/or A-3302-B.
FEBS Lett. 1990, 270 (1ꢀ2), 139–142.
S
Supporting Information. Materials and methods for
b
chemistry and isolation, Marfey's analysis, and NMR and MS
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
(13) Gorlero, M.; Wieczorek, R.; Adamala, K.; Giorgi, A.; Schinina,
M. E.; Stano, P.; Luisi, P. L. Ser-His catalyzes the formation of peptides
and PNAs. FEBS Lett. 2009, 583, 153–156.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 801-585-5234. Fax: 801-585-9119. E-mail: ews1@utah.edu.
(14) Marfey, P. Determination of D-amino acids. II. Use of a
bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res.
Commun. 1984, 49, 591–596.
(15) Shoji, J.; Hinoo, H.; Wakisaka, Y.; Koizumi, K.; Mayama, M.
Isolation of a new peptide antibiotic TL-119. Antibiotics from the genus
Bacillus. J. Antibiot. 1975, 28, 126–128.
(16) Ogawa, Y.; Mori, H.; Ichihashi, M.; Ueno, T.; Nakashima, T.;
Fukami, H.; Kakajima, R.; Ida, H. Structure elucidation of the antibiotics
A-3302-A and -B produced by a new strain of Bacillus subtilis. Pept. Chem.
1977, 123–126.
(17) Wenzel, S. C.; Meiser, P.; Binz, T. M.; Mahmud, T.; Mueller, R.
Nonribosomal peptide biosynthesis: point mutations and module skip-
ping lead to chemical diversity. Angew. Chem. 2006, 45, 2296–2301.
(18) Enomoto, M.; Kuwahara, S. Total synthesis of bacilosarcins A
and B. Angew. Chem. 2009, 48, 1144–1148.
(19) Thomas, K. C.; Sllabnis, A. S.; Johansen, M. E.; Lanza, D. L.;
Moos, P. J.; Yost, G. S.; Reilly, C. A. Transient receptor potential
vanilloid 1 agonists cause endoplasmic reticulum stress and cell death in
human lung cells. J. Pharmacol. Exp. Ther. 2007, 321, 830–838.
(20) Reilly, C. A.; Johansen, M. E.; Lanza, D. L.; Lee, J.; Lim, J.-O.;
Yost, G. S. Calcium-dependent and independent mechanisms of capsai-
cin receptor (TRPV1)-mediated cytokine production and cell death in
human bronchial epithelial cells. J. Biochem. Mol. Toxicol. 2005,
19, 266–275.
’ ACKNOWLEDGMENT
This work was funded by ICBG Grant U01TW008163 from
Fogarty (NIH) (E.W.S.), NIH Grant ES01734 (C.A.R.), and a
seed grant from the Department of Anesthesiology, University of
Utah (C.A.R.). We thank the government of the Philippines and
the community of Mactan Island for permission to conduct
this study.
’ ABBREVIATIONS USED
TRPV1 (VR1), transient receptor potential vanilloid-1; HEK-
239, human embryonic kidney cell line; CGRP, calcitonin gene-
related peptide; DRG, dorsal root ganglion; BEAS-2B, human
bronchial epithelial cell line; HRESIMS, high-resolution electro-
spray ionization mass spectrometry
’ REFERENCES
(1) Wu, L. J.; Sweet, T. B.; Clapham, D. E. International Union of
Basic and Clinical Pharmacology. LXXVI. Current progress in the
mammalian TRP ion channel family. Pharmacol. Rev. 2010, 62, 381–404.
(2) (a) Gazzieri, D.; Trevisani, M.; Springer, J.; Harrison, S.; Cottrell,
G. S.; Andre, E.; Nicoletti, P.; Massi, D.; Zecchi, S.; Nosi, D. Substance P
released by TRPV1-expressing neurons produces reactive oxygen spe-
cies that mediate ethanol-induced gastric injury. Free Radical Biol. Med.
2007, 43, 1670. (b) Caterina, M. J.; Schumacher, M. A.; Tominaga, M.;
Rosen, T. A.; Levine, J. D.; Julius, D. The capsaicin receptor: A heat-
activated ion channel in the pain pathway. Nature 1997, 389, 816–824.
(3) Szallasi, A.; Appendino, G. Vanilloid receptor TRPV1 antago-
nists as the next generation of painkillers. J. Med. Chem. 2004,
47, 2717–2723.
(21) Johansen, M. E.; Reilly, C. A.; Yost, G. S. TRPV1 antagonists
elevate cell surface populations of receptor protein and exacerbate
TRPV1-mediated toxicities in human lung epithelial cells. Toxicol. Sci.
2006, 89, 278–286.
(22) Reilly, C. A.; Taylor, J. L.; Lanza, D. L.; Carr, B. A.; Crouch,
D. J.; Yost, G. S. Capsaicinoids cause inflammation and epithelial cell
death through activation of vanilloid receptors. Toxicol. Sci. 2003,
73, 170–181.
(23) Chuang, H.; Lin, S. Oxidative challenges sensitize the capsaicin
receptor by covalent cysteine modification. Proc. Natl. Acad. Sci. 2009,
106, 20097–20102.
3754
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755

Journal of Medicinal Chemistry
ARTICLE
(24) Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. TRP
channel activation by reversible covalent modification. Proc. Natl. Acad.
Sci. 2006, 103, 19564–19568.
(25) Fernandez-Ballester, G.; Ferrer-Montiel, A. Molecular model-
ing of the full-length human TRPV1 channel in closed and desensitized
states. J. Membr. Biol. 2008, 223, 161–172.
(26) Miyamoto, T.; Dubin, A.; Petrus, M.; Patapoutian, A. TRPV1
and TRPA1 mediate peripheral nitric oxide-induced nociception in
mice. PloS ONE 2009, 4, e7596.
(27) Salazar, H.; Llorente, I.; Jara-Oseguera, A.; Garcia-Villegas, R.;
Munari, M.; Gordon, S.; Islas, L.; Rosenbaum, T. A single N-terminal
cysteine in TRPV1 determines activation by pungent compounds from
onion and garlic. Nature Neurosci. 2008, 11, 255–261.
(28) Susankova, K.; Tousova, K.; Vyklicky, L.; Teisinger, J.; Vlachova,
V. Reducing and oxidizing agents sensitize heat-activated vanilloid re-
ceptor (TRPV1) current. Mol. Pharmacol. 2006, 70, 383–394.
(29) Grandl, J.; Hu, H.; Bandell, M.; Bursulaya, B.; Schmidt, M.;
Petrus, M.; Patapoutian, A. Pore region of TRPV3 ion channel is
specifically required for heat activation. Nature Neurosci. 2008, 11,
1007–1013.
(30) Aneiros, E.; Cao, L.; Papakosta, M.; Stevens, E.; Phillips, S.;
Grimm, C. The biophysical and molecular basis of TRPV1 proton
gating. EMBO J. 2011, 30, 994–1002.
(31) Oseguera, A.; Islas, L.; Garcia-Villegas, R.; Rosenbaum, T. On
the mechanism of TBA block of the TRPV1 channel. Biophys. J. 2007,
92, 3901–3914.
(32) Friedman, M. Chemistry, nutrition, and microbiology of
D-amino acids. J. Agric. Food. Chem. 1999, 47, 3457–3479.
3755
dx.doi.org/10.1021/jm101621u |J. Med. Chem. 2011, 54, 3746–3755