Caspase-1 and -3 inhibiting drimane sesquiterpenoids from the extremophilic fungus Penicillium solitum

Article abstract of DOI:10.1021/np200528n

Two new drimane sesquiterpene lactones and one new tricarboxylic acid derivative were isolated from the Berkeley Pit extremophilic fungus Penicillium solitum. The structures of these compounds were deduced by spectroscopic analysis. Berkedrimanes A and B inhibited the signal transduction enzymes caspase-1 and caspase-3 and mitigated the production of interleukin 1-β in the induced THP-1 (pro-monocytic leukemia cell line) assay.

Full text of DOI:10.1021/np200528n

Note
pubs.acs.org/jnp
Caspase-1 and -3 Inhibiting Drimane Sesquiterpenoids from the
Extremophilic Fungus Penicillium solitum
,
†
,†
†
‡
‡
Donald B. Stierle,* Andrea A. Stierle,* Teri Girtsman, Kyle McIntyre, and Jesse Nichols
†
Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59812, United States
Department of Chemistry and Geochemistry, Montana Tech of the University of Montana, Butte, Montana 59701, United States
‡
*
S Supporting Information
ABSTRACT: Two new drimane sesquiterpene lactones and one new
tricarboxylic acid derivative were isolated from the Berkeley Pit extremophilic
fungus Penicillium solitum. The structures of these compounds were deduced by
spectroscopic analysis. Berkedrimanes A and B inhibited the signal transduction
enzymes caspase-1 and caspase-3 and mitigated the production of interleukin
1-β in the induced THP-1 (pro-monocytic leukemia cell line) assay.
icroorganisms isolated from the acidic, metal-rich waters
of the Berkeley Pit Lake System in Butte, Montana, have
The pathological hallmarks of Alzheimer’s disease include
M
neuronal loss, extracellular senile plaques containing the
10
been a rich source of interesting and biologically active
peptide amyloid-β, and neurofibrillary tangles (NFTs).
1
,2a−f
compounds.
We recently isolated a filamentous fungus
Although amyloid plaques and NFTs have been largely
regarded as independent neuropathologic phenomena, they
may be functionally linked, and caspases, particularly caspase-3
from surface waters of the Pit Lake, which was identified as
Penicillium solitum. The organic extracts of broth cultures of this
fungus exhibited potent activity in caspase-1, caspase-3, and
matrix metalloproteinase-3 enzyme inhibition assays. These
enzyme assays were used to guide the isolation of two new
drimane sesquiterpenes, berkedrimanes A and B. Their
isolation, characterization, inhibition of the caspases, and
mitigation of interleukin-1β production in induced THP-1
cells will be described in this report.
10
and -1, may play an important role in their development. We
have begun exploring the implications of compounds with
activity against both of these caspases. The development of new
enzyme inhibitors will not only provide potential chemo-
therapeutics or pharmacophore models but also provide tools
for the investigation of the intricacies of signal transduction.
P. solitum, a surface isolate of acidic Berkeley Pit Lake (pH 2.7),
was grown in mycological broth for 22 days. At time of harvest,
the mycelium was removed by filtration and the filtrate was
The role of caspase-1 in inflammation is complex. Caspase-1
is activated upon binding to the inflammasome, a multiprotein
complex that plays a key role in innate immunity by activating3
the proinflammatory cytokines interleukin 1-β and IL-18.
There is a strong correlation between dysregulated inflamma-
some activity and both inherited and acquired inflammatory
extracted with CHCl . When compared to other fungal extracts
3
from Pit microbes in our study, the organic extract of P. solitum
demonstrated excellent inhibitory activity in the signal
transduction assays. Flash silica gel chromatography followed
by silica gel HPLC yielded the two new drimane derivatives
1 and 2, which contributed to the observed enzyme inhibitory
activity. A known tricarboxylic acid (3) and its lactone (5) and
the new tricarboxylic acid derivative compound 7 were also
isolated from this extract.
3
diseases. Caspase-1 inhibitors have shown promise in either
delaying the onset or mitigating the effects of a number of
4
diseases, including Huntington’s disease, amyotropic lateral
5
6
7,8
sclerosis, stroke, and multiple sclerosis. Caspase-1 inhibitors
have also been proposed as potential therapies for osteoarthritis
9
10
and rheumatoid arthritis, Alzheimer’s disease, and brain and
nerve trauma.
The major drimane derivative, 1, isolated as a colorless oil,
6
,8
had a molecular formula of C H NO as deduced by
22 33
5
+
There is a complex relationship between different members
of the caspase enzyme class. Caspase-3 is an executioner
caspase and an important component of apoptosis, which can
HRCIMS [M + H] , with seven degrees of unsaturation. The
IR spectrum indicated the presence of an α,β-unsaturated
γ-butyrolactone and both ester and amide functionalities (1756,
11
be activated by either extrinsic or intrinsic factors. High levels
of caspase-3 have been associated with Alzheimer’s disease,
particularly when coupled with high levels of caspase-1.
Received: June 20, 2011
Published: January 25, 2012
©
2012 American Chemical Society and
American Society of Pharmacognosy
262
dx.doi.org/10.1021/np200528n | J. Nat. Prod. 2012, 75, 262−266

Journal of Natural Products
Note
correlations to methyl C-15 (δ 13.6) and olefinic C-7 (δ
C
C
1
35.7). H-7 (δ 6.88) exhibited three-bond correlations to C-9, -12,
H
and -5 (δ 44.3) connecting the B and C rings to the A ring. H-
C
5
(δ 1.74) exhibited three-bond correlations to C-1 (δ 75.5),
H C
C-15 (δ 13.6), and C-9 (δ 42.9). Oxygen-bearing methine
C
C
H-1 (δ 4.78) exhibited three-bond correlations to both valine
H
C-1′ (δ 171.5) and C-3 (δ 34.7), linking the tricyclic moiety
C
C
to valine. These data fit the drimane skeleton and generated the
structure of berkedrimane A (1) as shown.
Berkedrimane A (1) is isomeric with the known compound
1
3
purpuride, isolated from Penicillium purpurogenum (the
8
7
double bond is Δ rather than Δ ) and has the same
sesquiterpene skeleton as nebularilactone B isolated from
1
4
Lepista nebularis. Comparison of the NMR data of
berkedrimane A with those of nebularilactone B provided
additional support for the structure of compound 1 as shown.
The relative configuration of 1 was established by
consideration of coupling constants and from 1-D difference
NOE studies. H-1 appeared as a broad singlet, indicating that it
occupied an equatorial position in the cyclohexane ring and
that the ester substituent must therefore be axial. Irradiation of
H-9 (δ 3.18) enhanced the intensity of H-11α (4.23) as well
H
as that of H-5 (δ 1.74), indicating a 1,3-syn-diaxial relationship
H
between H-5 and -9. In C D the methyl singlets of H-13, H-14,
6
6
and H-15 shifted from δH 1.00, 0.99, and 0.92 ppm,
respectively, to δH 0.55, 0.66, and 0.27 ppm. This greater
signal dispersion in C D allowed selective irradiation of the
6
6
methyl resonances to observe the resultant NOE enhance-
ments. A large NOE enhancement was observed between
methyl H-14 and methyl H-15, indicating a 1,3-diaxial
relationship. Irradiation of H-15 also enhanced the absorption
of methine H-1. These data were sufficient to generate the
relative configuration of berkedrimane A (1), which was
consistent with that of purpuride and nebularilactone B.
The molecular formula of 2 was C H NO , established by
−
1
12
1
733, and 1675 cm , respectively). All 22 carbons were
1
3
observed in the C NMR spectrum, and analysis of this
spectrum along with the DEPT and HMQC spectra indicated
the presence of three ester/amide carbonyls (δ 171.5, 170.2,
69.7), one trisubstituted double bond (δ 135.7, 126.9), two
oxygen-bearing carbons (δ 75.5, 66.7), one nitrogen-bearing
carbon (δ 57.4), two quaternary carbons (δ 37.5, 33.0), three
methine carbons (δ 44.3, 42.9, 31.1), three methylene carbons
δ 34.7, 25.1, 22.7), and six methyl carbons (δ 32.7, 23.3,
1.5, 19.5, 17.3, 13.6). An N-acetyl valine residue was deduced
C
1
C
22 33
6
+
C
HRCIMS [M + H] . The IR spectrum again indicated the
presence of α,β-unsaturated γ-butyrolactone, ester, and amide
functionalities as in compound 1, but compound 2 also showed
C
C
C
−1
1
(
2
an additional −OH stretch at 3436 cm . The H NMR and
13
C
C
C NMR spectra (Table 1) for 1 and 2 were very similar, with
1
1
from the following data: the H− H COSY spectrum indicated
the following key exceptions: methine C-9 of compound 1 (δC
42.9) was replaced with a quaternary oxygen-bearing carbon in
the presence of the spin system NH−CH−CH−(CH ) , and
3
2
the HMBC spectrum showed a correlation from both NH and
compound 2 (δC 78.0); methylene H -11 in compound 1
2
H-2′ to carbonyl C-1′ (δ 171.5) and from NH and methyl
singlet H -2″ (δH 2.08) to carbonyl C-1″ (δ 170.2). These
appeared as two triplets (δ 4.23 and 4.02, J = 9.2 Hz), whereas
C
H
3
C
the analogous protons appeared as geminally coupled doublets
assignments accommodated C H NO and both the amide
at (δ 4.40 and 4.25, J = 10.1 Hz) in compound 2. These data
7
12
2
H
and the ester functionalities. There remained to assign
C H O , which suggested a sesquiterpene that included
suggested that the hydroxy moiety in 2 was at position C-9. All
of the HMBC correlations were similar to those of 1 and were
in accord with this proposed structure. Key NOE correlations
were also similar to those of 1, indicating the same relative
stereoconfiguration and the structure of berkedrimane B (2) as
shown.
15
21
3
three singlet methyls (δH 1.00, 0.99, 0.92) and the α,β-
unsaturated γ-butyrolactone, which accommodated three
degrees of unsaturation. The remaining two degrees required
two additional rings, suggesting a fused tricyclic carbon
skeleton.
3
The magnitude of the scalar coupling constants ( J) associ-
1
1
The H− H COSY spectrum indicated three isolated spin
ated with olefinic H-7 also supported the assigned configura-
tion at C-9. In the energy-minimized model (Molecular
Dynamics, MM2 force field calculations) of berkedrimane B
as shown, the dihedral angles of H-7 to H -6 and H -6 were
systems: OCH −CH, CH−CH −CH, and OCH−CH −
2
2
2
CH . The HMBC spectrum provided key correlations to
2
connect these systems and to assemble the molecule. The α,β-
unsaturated γ-butyrolactone provided an appropriate starting
α
β
−55° and 58°, respectively, which would indicate that H-7 was
a doublet of doublets (J = 4.0, 3.9). In the MM2 energy-
minimized model of 9-epi-berkedrimane, the dihedral angles
of H-7 were 100° relative to H -6 and −7.5° relative to H -6.
point. Oxygen-bearing methylene H-11α (δ 4.23) exhibited
H
three-bond correlations to olefinic C-8 (δ 126.9) and carbonyl
C
C-12 (δ 169.7), and H-11β (δ 4.02) had a similar correlation
C
H
β
α
to C-10 (δ 37.5). Both protons showed two-bond correlations
If this were the case, H-7 would be a doublet of doublets
C
to C-9 (δC 42.9). H-9 (δH 3.18) showed three-bond
(J = 12, 1). In fact, H-7 is a triplet (J = 3.5), which supports the
2
63
dx.doi.org/10.1021/np200528n | J. Nat. Prod. 2012, 75, 262−266

Journal of Natural Products
Note
Table 1. 13C and H NMR Data for Compounds 1, 2, and 8 in CDCl3
1
a
1
2
8
d
b
d
b
d
c
no.
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
5.01, bs
δC, mult.
δH, mult. (J in Hz)
1
2
75.5, CH
22.7, CH2
4.78, bs
1.85, m
76.3, CH
23.5, CH2
173.4, C
1.92, dt (14.5, 3.1)
1.76, dq (14.5, 3.5)
1.61, td (13.7, 3.4)
1.35, dt (13.7, 3.7)
28.6, CH2
2.46, m
1
.76, m
1.69, m
.32, dt (13.7, 3.2)
2.11, m
2.17
3
34.7, CH2
34.8, CH2
32.3, CH2
1
1.97
4
5
6
33.0, C
32.6, C
77.7, C
53.8
44.3, CH
25.1, CH2
1.74, dd (11.5, 6.0)
2.50, m
37.9, CH
25.5, CH2
2.51, dd (15.3, 5.8)
2.76
2.58, ddd (20.1, 5.7, 3.9)
2.24, m
27.7, CH2
1.82, m
1.35, m
1.22, bs
1.22, bs
1.22, bs
1.22, bs
1.22, bs
2
.21, m
7
8
9
1
1
135.7, CH
126.9, C
42.9, CH
37.5, C
6.88, bq (3.5)
139.9, CH
130.4, C
78.0, C
7.01, t (3.5)
27.9, CH2
29.5, CH2
29.3, CH2
29.5, CH2
29.3, CH2
3.18, m
0
1
41.0, C
66.7, CH2
α 4.23, t (9.2)
β 4.02, t (9.2)
73.9, CH2
4.40, d (10.1)
4.25, d (10.1)
1
2
3
169.7, C
168.5, C
31.9, CH2
33.8, CH2
1.22, bs
2.03, bq
1
32.7, CH3
1.00, s
33.0, CH3
1.04, s
1
.95, m
1
4
5
21.5, CH3
13.6, CH3
0.98, s
0.92, s
21.6, CH3
18.6, CH3
1.03, s
0.92, s
139.2, CH
114.1, CH2
5.78, ddt (17.1, 10.2, 6.8)
4.91, ddt (10.2, 2.0, 1.2)
1
4
.97, ddt (17.1, 2.0, 1.6)
1
1
1
2
3
4
5
6
7
′
174.4, C
174.4, C
51.9, CH3
51.8, CH3
53.1 CH3
171.5, C
170.9, C
3.64, s
3.79, s
3.70, s
′
57.4, CH
31.1, CH
19.5, CH3
17.3, CH3
170.2, C
4.62, m
58.9, CH
29.9, CH
19.4, CH3
17.9, CH3
169.9, C
4.36, dd (7.4, 5.8)
2.20, m
′
2.21, m
′
1.02, d (6.8)
0.93, d (7.0)
1.02, d (7.0)
0.98, d (6.8)
′
Ac
Ac CH3
NH
23.3, CH3
2.08, s
23.2, CH3
2.07, s
5.90, bd (8.4)
5.94, bd (6.8)
a
b
1
c
1
All assignments are based on COSY, HSQC, HMBC, and NOE experiments. 300 MHz for H NMR, HMBC, COSY. 500 MHz for H NMR,
d
13
HMBC, COSY. 75 MHz for C NMR.
+
structure of compound 2 as shown. Similar results were obtained
when the minimum energy geometries were determined using
Hartree−Fock (3-21G) calculations for compound 2 and its
C-9 epimer.
The HRCIMS [M + H] of the trimethyl ester 8 established
1
13
a molecular formula of C20H O . The H and C NMR
spectra of compounds 4 and 8 were almost identical, with one
key exception; the H NMR of trimethyl ester 4 indicated the
presence of a terminal methyl group, and this methyl triplet was
not present in the H NMR spectrum of compound 8. Instead,
both the H and C NMR spectra indicated the presence of a
terminal monosubstituted double bond [δ 114.1, δ 4.97 (ddt,
J = 17.1, 2.0, 1.6), 4.91 (ddt, J = 10.2, 2.0, 1.2) and δ 139.2, δH
34 7
1
The absolute configuration of the valine residue in
1
15
berkedrimane B (2) was established by Marfey’s analysis.
1
13
Compound 2 was hydrolyzed in boiling 6 M HCl and treated
with 2,4-dinitro-5-fluoro-L-alanamide (Marfey’s reagent). The
resulting derivative was analyzed by LC-MS. Authentic samples
of D- and L-valine were derivatized under identical conditions.
The valine derivative from the hydrolysis of 2 was identical to
the L-valine stereoisomer.
C
H
C
5
.78 (ddt, J = 17.1, 10.2, 6.7)]. This placed the double bond at
the end of the straight chain and established the structure of
triacid 7 as the previously undescribed 4-hydroxy-4,5-dicarboxy-
1
4-pentadecenoic acid.
We also isolated two tricarboxylic acids and a tricarboxylic
acid lactone along with the two drimane derivatives. These
acids were difficult to purify, but were easily separated following
methylation. A mixture of acids 3, 5, and 7 was treated with
diazomethane and separated by HPLC to yield the methyl
esters 4, 6, and 8. Analysis of NMR and mass spectral data
indicated that compound 4 was the methyl ester of
Secospiculisporic acid 3 and spiculisporic acid 5 have
received much attention because of their amphiphilic nature
and their ability to aggregate as liposome-like vesicles. They act
as biosoaps and biosurfactants and can associate with metals in
solution and precipitate these metal salts. According to the
NCBI PubChem Web site, spiculosporic acid was evaluated in
3 different biological assays, including several enzyme assays,
and was inactive in each test.
Berkedrimanes A and B were evaluated for their ability to
inhibit caspase-1 and caspase-3 in vitro in fluorometric assays.
Berkedrimane A (1) exhibited an IC value of 100 μM against
18
6
16
19
secospiculisporic acid (3), and compound 6 was the methyl
17
ester of the tricarboxylic acid lactone, spiculisporic acid (5).
Both of these acids have been previously described as
17
metabolites of Penicillium spiculisporum.
50
2
64
dx.doi.org/10.1021/np200528n | J. Nat. Prod. 2012, 75, 262−266

Journal of Natural Products
Note
300 or 500 MHz, and the 13C NMR spectra were recorded at 75 MHz
unless otherwise noted. All of the chemical shifts were recorded with
caspase-1 and 200 μM against caspase-3. It maintained
moderate inhibition of caspase-3 with an IC25 value of 15 μM.
Berkedrimane B (2) was slightly more potent, with IC values
respect to the deuterated solvent shift (CDCl , δH 7.24, δC 77.0 or
3
50
C D , δ 7.18, δC 128.0). Mass spectral data were provided by the
6
6
H
of 90 μM against caspase-1 and 50 μM against caspase-3. It
University of Montana Mass Spectrometer Facility. All solvents used
were spectral grade or distilled prior to use.
maintained moderate inhibition of caspase-3 with an IC value
25
of 10 μM. The three methyl esters were inactive against the two
caspases and MMP-3.
Collection, Extraction, and Isolation Procedures. The
collection and isolation of Berkeley Pit fungi have previously been
2
e
described. Penicillium solitum was grown in potato dextrose broth
8 flasks with 500 mL of broth per flask, in 1 L Erlenmeyer flasks) for
2 days (still culture). At time of harvest MeOH (50 mL per flask) was
(
2
added to each flask, the mycelium was removed by filtration, and the
filtrate was extracted with CHCl (3 × 1 L). Removal of the CHCl in
3
3
vacuo yielded 0.4128 g of total crude extract. This extract showed
potent enzyme inhibition in the MMP-3, caspase-1, and caspase-3
assays. It was chromatographed on a flash Si gel column in a gradient
fashion, starting with hexane, then with increasing amounts of IPA.
The column was washed with 100% IPA and finally with MeOH. The
fraction that eluted with 50% IPA/hexane was active in both the
caspase-1 and caspase-3 enzyme inhibition assays. It was further
purified with Si gel HPLC in an isocratic mode using 20% IPA/hexane
to yield berkedrimanes A (1) (11.6 mg) and B (2) (5.1 mg). The two
berkedrimanes, which were 2.8% and 1.2% of the crude CHCl3,
respectively, were active in the enzyme inhibition assays, although
several of the other column fractions exhibited activity as well. The
fraction that eluted with 20% IPA (180.2 mg) was a mixture of acids
(3, 5, and 7) that was difficult to separate. The acid fraction was
methylated with diazomethane and then chromatographed on Sigel
HPLC to give methyl esters of the known 4-hydroxy-4,5-di-
carboxypentadecanoic acid (3) (40.3 mg), the previously undescribed
Figure 1. Key HMBC correlations for berkedrimane 1.
When induced with titanium nanowires and bacterial
lipopolysaccharide (LPS), THP-1 cells produce large numbers
of inflammasomes, which in turn activate pro-IL-1B to the
active form, IL-1β. Induced THP-1 cells were exposed to
compounds 1 and 2 at concentrations of 100, 10, and 1 μM,
and the concentrations of IL-1β postexposure were determined
(
Figure 2). Both berkedrimanes effectively inhibited the
4
-hydroxy-4,5-dicarboxy-9-pentadecenoic acid (7) (75.3 mg), and
spiculisporic acid (5) (60.2 mg).
Berkedrimane 1: colorless oil, [α]25 +31.4 (c 0.0014, CHCl ); IR
D
3
(
CHCl ) ν 3436, 2966, 1756, 1733, 1675, 1506, 1373, 1016, 892
3 max
−1
1
13
1
cm ; H NMR and C NMR (CDCl ) see Table 1; H NMR (C D ,
3
6
6
5
0
1
00 MHz) δ 0.27 (s, 3H, H-15) 0.55 (s, 3H, H-13) 0.66 (s, 3H, H-14)
.82 (d, J = 6.73 Hz, 6H, H-4′,5′) 1.50 (s, 3H, OAc) 1.89 - 1.99 (m,
H) 3.11 (m, 1H, H-9) 3.60 (m, 1H, H-11) 4.19 (m, 1H, H-11) 4.56
(m, 1H, H-1) 4.66 (m, 1H, H-2′) 5.32 (bd, 1H, NH) 6.61 (m, 1H,
+
H-7); HRCIMS [M + H] m/z 392.2466 (calcd for C H NO ,
2
2
34
5
3
92.2437).
Berkedrimane 2: colorless oil, [α]25 −15.3 (c 0.0145, CHCl ); IR
D
3
(CHCl ) ν 3446, 3081, 2966, 1751, 1730, 1670, 1238, 1022, 729
3 max
−1
1
13
cm ; H NMR (CDCl ) see Table 1; C NMR (CDCl ) see Table 1;
EIMS m/z [M] 407 (2), 390 (10), 230 (55), 78 (100); HRCIMS
Figure 2. Production of IL-1β in THP-1 cells induced with TiO and
3
3
2
+
bacterial LPS following treatment with compounds 1 and 2.
+
[
M + H] m/z 408.2361 (calcd for C H NO , 408.2386).
22 33 6
Determination of the Absolute Configuration of Valine.
Drimane 2 (1 mg) was suspended in freshly distilled, constant boiling
M HCl (100 μL) and heated in a Teflon-sealed glass vial at 110 °C
for 15 h. The solution was then concentrated to dryness under a
production of IL-1β by intact inflammasomes at low micro-
molar concentrations, showing greater potency in the cell line
assay than in the enzyme assay. Further work is under way to
determine the relevance of these observations.
6
stream of N . This solid was treated with a solution of 2,4-dinitro-5-
2
The inflammasome is becoming an important therapeutic
target as studies continue to link inflammation and disease. In
mice, potential therapeutic agents that target the inflammasome
and mitigate the production of IL-1β have been shown to
reduce the inflammatory response following thromboembolic
fluoro-L-alanamide (100 μL, 1% w/v in acetone), followed by 1.0 M
NaHCO (25 μL), and heated in a Teflon-sealed glass vial at 80 °C for
3
10 min. The mixture was cooled and quenched with 1.0 M HCl
(
25 μL). Authentic L- and D-valine standards were derivatized under
15
identical conditions.
2
0
LC-MS Analysis. The Marfey’s derivatives and the standards
derivatives (see above) were analyzed by LC-MS using a ZORBAX
Extend C-18 (2.1 × 50 mm, 3.5 μM) column. Chromatography
stroke and to improve histopathology following traumatic
2
1
brain injury. The biological activity of the berkedrimanes is
similar to that of parthenolide, an anti-inflammatory
sesquiterpene lactone isolated from feverfew, Tanacetum
parameters are as follows: initial, 80% solvent A (H O + 0.1% formic
2
acid), 20% solvent B (acetonitrile), at 9 min to 60% A, at 10 min to
100% B. PDA parameters were as follows: channel A 340 nm, channel B
2
2
parthenium.
2
54 nm. MS parameters were as follows: APCI source. Retention time
for the authentic L-valine derivative was 10.6 min; retention time for
the authentic D-valine was 12.6 min. The Marfey’s derivative of the
hydrolysis of 2 had a retention time of 10.6 min and gave a mass
spectrum identical to the authentic valine samples.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded on a Perkin-Elmer 241 polarimeter using a 1 mL cell. IR
spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer.
Methylation of Carboxylic Acid Mixture. The mixture of acids
(10.3 mg) was dissolved in diethyl ether (2 mL), and a solution of
CH N dissolved in ether was added dropwise until the yellow color
1
13
H NMR and C NMR spectra were obtained on Bruker DPX-300 or
1
Varian 500 NMR spectrometers. H NMR spectra were recorded at
2
2
2
65
dx.doi.org/10.1021/np200528n | J. Nat. Prod. 2012, 75, 262−266

Journal of Natural Products
Note
remained. After 1 min the solvent was removed in vacuo and the
mixture subjected to HPLC using 5% EtOAc/hexane. The trimethyl
ester of the known tricarboxylic acid, secospiculisporic acid (4-hydroxy-
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3
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(
m, 2H) 2.46 (m, 1H) 2.76 (dd, J = 11.8, 3.3 Hz, 1H) 3.64 (s, 3H)
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(
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+
H] m/z 387.2344 (calcd for C H O , 387.2383).
20
35
7
Signal Transduction Assays. The signal transduction drug
discovery kits for caspase-1, caspase-3, and matrix metalloproteinase-3
enzymes were purchased from Enzo Life Sciences. The positive controls
1
(
(enzyme inhibitors) used in these assays are Ac-YVAD-CHO for the
caspase-1 assay, Ac-DEVD-CHO for the caspase-3 assay, and N-isobutyl-
N-(4-methoxyphenylsulfonyl)glycylhydroxamic acid for the MMP-3 assay.
In Vitro THP-1 Assay. Human monocyte cell line THP-1 was
(
(
purchased from ATCC (#TIB-202). The cells were suspended at
5
(
2−4) × 10 viable cells/mL in RPMI media supplemented with 10% fetal
(
bovine serum, 0.05 mM 2-mercaptoethanol, sodium pyruvate, and an
antimycotic/antibiotic cocktail containing penicillin, streptomycin, and
amphotericin B (Mediatech, VWR). The cells were differentiated into
macrophage-like cells by the phorbol ester PMA (1 μg/mL, Sigma)
(
2
4 h prior to experimentation. The transformed cells were removed from
the flask by scraping and centrifuged at 450g for 5 min. The resulting
cell pellet was suspended at 1.0 × 10 cells/mL and exposed to
6
(
caspase-1 inhibitors at concentrations described below (0.5−0.005%),
LPS [20 ng/mL], and TiO nanowires (100 μg/mL). Experiments
2
(
were conducted in 96-well plates for 24 h in 37 °C water-jacketed CO2
incubators (ThermoForma).
(
Toxicity Assay. Cell viability was determined by MTS reagent
using the CellTiter96 assay (Promega), according to the manufac-
turer’s protocol. The plate was read at 490 nm.
Cytokine Assays. Human IL-1β DuoSet was obtained from R&D
Systems, and ELISA assays were performed according to the
manufacturer’s protocol. The plate was read at 490 nm.
(
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(
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(
*
S
Supporting Information
Experimental details including the 1H NMR spectrum of
1
(
berkedrimane A (1) in both CDCl and C D and of berkedrimane
3
6 6
13
B (2) in CDCl , the C NMR, COSY, HMBC, and HSQC spectra
for both compounds in CDCl , and the H NMR and C NMR
3
sids&reqid=6542588476838064189.
1
13
3
(20) Abulafia, D. P.; de Rivero Vaccari, J. P.; Lozano, J. D.; Lotocki,
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spectra of trimethyl ester 8 in CDCl . This material is available free
3
of charge via the Internet at http://pubs.acs.org.
(
21) de Rivero Vaccari, J. P.; Lotocki, G.; Alonso, O. F.; Bramlett, H. M.;
Dietrich, W. D.; Keane, R. W. J. Cereb. Blood Flow Metab. 2009, 29,
AUTHOR INFORMATION
■
*
1251−1261.
(22) Juliana, C.; Fernandes-Alnemri, T.; Wu, J.; Datta, P.; Solorzano,
Corresponding Author
Tel: 406-243-2094. E-mail: donald.stierle@mso.umt.edu;
andrea.stierle@mso.umt.edu.
L.; Yu, J.; Meng, R.; Quong, A. A.; Latz, E.; Scott, C. P.; Alnemri, E. S.
J. Biol. Chem. 2010, 285, 9792−9802.
ACKNOWLEDGMENTS
■
We thank B. Parker (University of Montana) for high-
resolution mass spectrometry data. We thank NSF grant
CHE-9977213 for acquisition of an NMR spectrometer and the
MJ Murdock Charitable Trust ref 99009:JVZ:11/18/99 for
acquisition of the mass spectrometer. The project was
supported by NIH grants R01CA139159, P20RR16455-04,
P20RR017670, 5P30NS055022, and RC2ES018742.
2
66
dx.doi.org/10.1021/np200528n | J. Nat. Prod. 2012, 75, 262−266