International Immunopharmacology 15 (2013) 532–538

Contents lists available at SciVerse ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

In vitro anti-allergic activity of the fungal metabolite pyridovericin

Marcela de Souza Santos a, b, Willian Jonis Andrioli c, Maria Perpétua Freire de Morais Del Lama a, b,
Jairo Kenupp Bastos c, N.P. Dhammika Nanayakkara d, Rose Mary Zumstein Georgetto Naal a, b,⁎
a
Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto. Avenida do Café, s/n. Ribeirão Preto 14040-903, São Paulo, Brazil
b
Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas 13083-970, Brazil
c
Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto. Avenida do Café, s/n. Ribeirão Preto 14040-903, São Paulo, Brazil
d
National Center of Natural Products Research, University of Mississippi, Oxford 38677, MS, USA

a r t i c l e i n f o a b s t r a c t

Article history: Mast cells play a critical role during the development of an allergic response. Upon activation by an antigen
Received 31 October 2012 and IgE, via FcεRI receptors, mast cells release histamine and other mediators that initiate and propagate
Received in revised form 9 January 2013 immediate hypersensitivity reactions. Mast cells also secrete cytokines that regulate the immune responses.
Accepted 23 January 2013
In this way, inhibitors of mast cell activity could work as promising therapeutics for allergic disorders. In
Available online 9 February 2013
the present work, we investigated the capacity of pyridovericin, a natural product isolated from the
Keywords:
entomopathogenic fungus Beauveria bassiana, to inhibit mast cell degranulation and cytokine secretion. It
Allergy was found that pyridovericin strongly decreased the release of β-hexosaminidase, a marker for mast cell de-
Ca2 + influx granulation, when mast cells were stimulated by both FcεRI-dependent and independent pathways. In addi-
Interleukin-4 tion, pyridovericin strongly abrogated secretion of interleukin-4. Pyridovericin-mediated suppression of
Mast cell degranulation stimulated increase in intracellular Ca2+ levels, a crucial signal for mounting of both degranulation and cyto-
Pyridovericin kine production responses, was ascribed as one of the inhibition targets of pyridovericin. Those initial studies
identify pyridovericin as a potential new candidate for the development of new anti-allergic drugs.
© 2013 Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction endoplasmic reticulum (ER) to trigger the release of calcium (Ca2+)

The Western world has faced an epidemic of respiratory allergies over
the last five decades [1]. It has been estimated that 10–20% of the world
population suffer from allergic rhinitis [2], in addition to the existence of
about 300 million asthmatic patients, a number that might have escalated
to 400 million by 2025 [3]. Allergic inflammation is mediated by the ex-
pansion of the T helper 2-subset (Th2) of T cells and isotype switching
of B cells to generate IgE antibodies towards specific allergens [4]. IgE, in
turn, binds tightly to its immune receptor, FcεRI, localized at the surface
of mast cells [5]. Antigen-induced cross-linking of mast cell IgE–FcεRI
complexes initiates Lyn kinase-mediated tyrosine phosphorylation of
FcεRI ITAMs (Immunoreceptor tyrosine-based activation motif), resulting
in recruitment and activation of Syk tyrosine kinase that, in turn,
phosphorylates several downstream targets, including phospholipase C
γ (PLCγ). This activates PLCγ to generate inositol 1,4,5-triphosphate
(IP3) and diacylglicerol (DAG) as products of phosphatidylinositol
4,5-biphosphate hydrolysis [6]. IP3 binds to its receptor at the
⁎ Corresponding author at: Departamento de Física e Química, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto. Avenida do Café, s/n. Ribeirão Preto 14040-903, São
Paulo, Brazil. Tel.: +55 1636024229; fax: +55 1636024880.
E-mail addresses: marcelafarmausp77@gmail.com (M. de Souza Santos),
andrioliw@yahoo.com.br (W. Jonis Andrioli), mpemdel@fcfrp.usp.br
(M.P. Freire de Morais Del Lama), jkbastos@fcfrp.usp.br (J. Kenupp Bastos),
dhammika@olemiss.edu (N.P.D. Nanayakkara), rmzgnaal@fcfrp.usp.br
(R.M. Zumstein Georgetto Naal).
from ER stores, which activates store-operated Ca2+ entry (SOCE),
whereas DAG participates with Ca2+ mobilization to activate protein ki-
nase C [7]. These signals synergize to activate mast cell degranulation
for release of a number of preformed allergic mediators, as well as for
the de novo synthesis and secretion of various lipid mediators and cyto-
kines that, collectively, mediate the early and late phases of allergic reac-
tions [5,8]. In this way, mast cells have been widely regarded as the key
players during the development of allergic reactions [9–14], and, there-
fore, inhibitors of mast cell activation and secretion of allergic mediators
are promising targets for therapeutic intervention.
Natural products have played a highly significant role in drug de-
velopment over the past century [15]. The purposeful design of natu-
ral products – aimed to increase the survival of the producer
organism in its ecological niche – coupled with abundant scaffold di-
versity, confer great advantage to the use of natural products as a
source of drug candidates [15]. The continuing role of natural prod-
ucts in the expansion of the therapeutic armamentarium, highlighted
by Newman and Cragg [16], can be largely attributed to the pharma-
cological activity of metabolites originated from microorganisms. In
this context, fungi appear as producers of secondary metabolites of
great contribution to the pharmaceutical sciences, taking as examples
penicillin, cyclosporine and statins [17]. Analysis of ca. 1500 fungal
metabolites, isolated between 1993 and 2001, showed that more
than 50% of these compounds exhibited antibacterial, antifungal or
antitumor actions [18].

1567-5769 © 2013 Elsevier B.V. Open access under the Elsevier OA license.
http://dx.doi.org/10.1016/j.intimp.2013.01.017
 M. de Souza Santos et al. / International Immunopharmacology 15 (2013) 532–538
Entomopathogenic fungi have emerged as a promising source of bi-
ologically active secondary metabolites [19]. It is presumed that many
entomopathogenic fungi lead to death of its host by inhibiting its en-
zymes and interfering with its regulatory system through the produc-
tion of toxic metabolites [20]. The entomopathogenic fungus Beauveria
bassiana has as the main component of its mycelial cake, the alkaloid
4-hydroxy-2-pyridone known as pyridovericin. The isolation and
characterization of pyridovericin were carried out in 1998 by Takahashi
et al. [19]. This metabolite comprises 4-hydroxy-5-(ρ-hydroxyphenyl)
pyridine and 4-methyl-octahydro-6-hydroxymethyl-2,4-dienoil moie-
ties, in addition to a chiral carbon at position 12 with R configuration
(Fig. 1). The 4-hydroxy-2-pyridone alkaloids belong to a family of natu-
ral products with great chemical diversity and biological activity, the
latter including antifungal, antibacterial, insecticidal and cytotoxic ac-
tivities [21–23]. Although chemically well characterized, the biological
activity of pyridovericin reported so far includes only a weak inhibitory
effect of tyrosine kinases from NIH3T3/v-src cells [20]. The poorly ex-
plored pharmacological usability of pyridovericin prompted us to inves-
tigate the capabilities of this metabolite to inhibit antigen-stimulated
activation of mast cells, as an initial investigation that could, ultimately,
demonstrate the application of pyridovericin as a new anti-allergic
candidate.
2. Material and methods
2.1. Fungal material
The entomopathogenic fungus was isolated from a soil sample col-
lected in Crato, Ceará Province, Brazil (7° 14′2″ S, 39° 24′32″ W), on
January 2006. The isolate was identified as B. bassiana based on its
morphology and a sequence analysis of the ITS region of its rDNA
(GenBank accession number HQ665468). A voucher specimen (num-
ber 4408) is deposited at the Department of Mycology of the Federal
University of Pernambuco.
2.2. Culture and isolation of pyridovericin
The fungus B. bassiana was inoculated onto agar plates [24] and in-
cubated at 30 °C for 7 days. Three agar plugs (0.5 cm diameter) were
cut and inoculated in 50 Erlenmeyer flasks containing 90 g of solid me-
dium each (rice–oat), and were maintained at 30 °C, for 60 days. After
this period, the mycelia mass was macerated with ethanol overnight
and filtered. The filtrate was concentrated under vacuum to furnish
the crude ethanolic extract (18 g), which was partitioned sequentially
with equal volumes of n-hexane and ethyl acetate (EtOAc) three
times each, yielding both hexane (5.3 g) and EtOAc fractions (9.3 g).
The EtOAc fraction was chromatographed on Sephadex LH-20 column
(4 cm× 60 cm, eluted with MeOH), providing eight fractions. Fraction
B6 (562 mg) was subject to column chromatography on silica gel
(3 cm× 25 cm, with gradient elution composed of increasing mixtures
of CH2Cl2 and CH3OH), providing eight fractions. Subfraction B6-7
(81 mg) was submitted to preparative TLC using CH2Cl2–CH3OH (9:1,
v/v) as eluent to afford pyridovericin (40 mg) as the major compound.
The purity was verified by analytical HPLC (C18 reverse-phase column)
Fig. 1. Chemical structure of pyridovericin. CAS RN 210976-24-02.
533
using CH3CN–H2O (with gradient elution, from 10% to 100% of CH3CN)
at flow rate of 1.0 mL.min−1 over 25 minute-period.
2.3. Pyridovericin
Pale yellow powder; mp 201–204 °C; [α]D20 −15.0 (c 0.3, M
CH3OH); UV (CH3OH) λmax 215 (2.73), 245 (2.56), 340 (2.38) nm;
IR νmax 3470, 3373, 3100, 1679, 1604, 1471 992 cm −1; for 1H NMR
and 13C NMR spectroscopic data, see Supplementary Materials;
HREIMS (ESI–TOF) m/z 370.1646 [M + H] + (calcd for C21H23NO5,
370.1649).
2.4. Cell culture
RBL-2H3 cells [25] were maintained in monolayer culture in mini-
mum essential medium with L-glutamine and Eagle's salt, supplemented
with 20% fetal bovine serum (Atlanta Biological, Atlanta, USA) and
50 μg/mL gentamicin sulfate (Invitrogen Corp., Carlsbad, USA). The
cells were used from 3 to 5 days after passage, and released from culture
flasks by treatment with 0.5% trypsin-EDTA for 5 min at 37 °C,
centrifuged at 1000 ×g for 5 min, and resuspended at 1× 106 cell/mL.
Unless otherwise specified, reagents were from Gibco (Carlsbad, USA).
2.5. Degranulation assay
IgE-mediated degranulation of mast cells was accessed as described
by Naal and others [26]. RBL-2H3 cells were initially sensitized with
1 μg/mL anti-DNP IgE (purified as previously described, [27]) and plated
onto 96-well plate at 5 × 10 5 cell/mL for 24 h, at 37 °C and 5% CO2 atmo-
sphere. Next, cells were washed twice in Tyrode's buffer (135 mM NaCl,
5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 20 mM HEPES,
and 1 mg/mL bovine serum albumin (BSA) at pH 7.4) and pretreated
with different concentrations of pyridovericin (0.1–100 μM, from a
DMSO stock solution), diluted in Tyrode's buffer, for 20 min. Cell de-
granulation was then stimulated by addition of 100 μL of either
100 ng/mL DNP-BSA (BSA conjugate with an average of 15 DNP groups,
prepared as previously described, [28]) or 200 nM thapsigargin
(Sigma-Aldrich, St. Louis, USA), both diluted in Tyrode's buffer. Samples
were then incubated for 1 h at 37 °C, after which, degranulation was
stopped by placing cells on ice. Cell degranulation was measured by de-
termining the amount of released β-hexosaminidase. In this way, 25 μL
of cell supernatant and 100 μL of 1.2 mM β-hexosaminidase
substrate (4-methylumbelliferyl-N-acetyl-β-D-glucosaminide, MUG,
Sigma-Aldrich), in 0.05 M sodium acetate buffer (pH 4.4), were mixed
in separate 96-well plate and incubated for 30 min, at 37 °C. The en-
zyme–substrate reaction was stopped by addition of 175 μL of 0.1 M
carbonate buffer, pH 10. Controls without antigen were used to mea-
sure spontaneous release. Total β-hexosaminidase release was obtained
by lysing cells with 0.1% Triton-X 100 prior to supernatant removal.
Released β-hexosaminidase was quantified by measuring the fluores-
cence intensity of the product of β-hex-mediated cleavage of MUG,
methylumbelliferone, in a microplate reader (BioTEK, Winooski, USA)
using 360 nm excitation and 450 nm emission filters. The percentage
of stimulated β-hexosaminidase-release was calculated according to
Eq. (1). Values of IC50 (concentration of pyridovericin necessary to in-
hibit the release of β-hexosaminidase from cells stimulated with anti-
gen by 50%) were determined graphically. Unless otherwise specified,
reagents were from Mallinckrodt Baker Inc., Phillipsburg, USA.
 
S−N
% β‐hexosaminidase ¼  100 ð1Þ
T−N
N Normal, fluorescence from vehicle (Tyrode's buffer);
S Sample, fluorescence from (+) DNP-BSA/thapsigargin, (+)
or (−) pyridovericin samples
T Total, fluorescence from Triton X-100-lysed cell samples.
534 M. de Souza Santos et al. / International Immunopharmacology 15 (2013) 532–538
2.6. β-Hexosaminidase activity assay
RBL-2H3 cell suspension, at 5 × 10 5 cell/mL, was sonicated during
20 min for cell lysis and release of β-hexosaminidase enzyme. After cen-
trifugation at 1000 ×g for 5 min, 45 μL of supernatant was incubated
with 5 μL of pyridovericin sample solutions (0.01–100 μM) and 50 μL
of β-hexosaminidase substrate (MUG) solution (final concentration
1.2 mM), for 60 min. The enzyme–substrate reaction was stopped by
addition of 200 μL of stop solution (0.1 M carbonate buffer, pH 10.0).
β-Hexosaminidase activity was determined from fluorescence mea-
surements of hydrolyzed MUG, as described above. Samples not treated
with pyridovericin were taken as 100% of β-hexosaminidase activity.
2.7. Ca 2+ measurements
Cytoplasmic Ca 2+ levels were measured using a steady-state fluo-
rimeter, (Hitachi F-4500, Kyoto, Japan). RBL-2H3 cells suspended at
4 × 106 cell/mL in Tyrode's buffer were loaded with the Ca2+ indicator
Fluo-4 AM (0.5 μM; Invitrogen; excitation 490 nm, emission 520 nm)
and sensitized with 1 μg/mL anti-DNP IgE in Tyrode's buffer containing
0.5 mM sulfinpyrazone (Sigma-Aldrich) and 1 mg/mL BSA for 1 h at
37 °C. Following a washing step with Tyrode's buffer, cells were
resuspended at 2 × 106 cell/mL using the same buffer. In the fluorime-
ter, the cell suspension was kept at 37 °C and continuously stirred for
following pretreatment with 10 μM pyridovericin, for 20 min, after
which cells were stimulated with either 0.4 μg/mL DNP-BSA or
200 nM thapsigargin.
2.8. Quantification of IL-4 secretion
RBL-2H3 cells, plated at 1×105 cell/mL, were sensitized with 1 μg/mL
anti-DNP IgE and cultivated overnight. Next, cells were incubated with
different concentrations of pyridovericin and 0.4 μg/mL DNP-BSA for
4 h at 37 °C. Supernatants were collected and absolute amounts of IL-4
released by cells were determined using a mouse IL-4 immunoassay sys-
tem (BD Biosystems, Franklin Lakes, USA).
2.9. Cell viability assay
RBL-2H3 cells were seeded onto 96-well microplates at 3.5× 105 and
2.5 × 105 cell/mL, corresponding to 1 and 4 h cell-pyridovericin incuba-
tion times, respectively, and incubated overnight at 37 °C. After a wash-
ing step with PBS, cells were treated with different concentrations of
pyridovericin (0.1–100 μM), diluted in cell culture media, following
an incubation step for 1 or 4 h, at 37 °C. Next, samples were added
with 5 μL of a 10 mg/mL resazurin stock solution (Sigma-Aldrich) for
new 3 h incubation. Cell viability was assessed based on fluorescence
measurements of resorufin, a fluorescent product resulting from reduc-
tion of resazurin by viable cells (excitation 530 nm, emission 390 nm).
Fluorescence registered from non-treated control samples was taken as
100% of cell viability.
2.10. Statistical analysis
Results were expressed as mean ± SD. Statistical significance was
assessed by one-way ANOVA followed by Dunnett's posthoc pairwise
comparisons. Differences were considered statistically significant at
*p b 0.05, **p b 0.01, and ***p b 0.001.
3. Results
We first evaluated the effects of pyridovericin on stimulated de-
granulation of RBL-2H3 cells, an immortal cell line derived from a
rat leukemia composed of basophil-like cells [29,30]. Among the
three major histamine-containing rodent cells, i.e., basophils, mucosal
and connective tissue mast cells, RBL cells were previously reported
to closely resemble rodent mucosal mast cells and basophils, but to
share a smaller number of morphological and functional features
with rodent connective tissue mast cells [30]. For instance, basophils,
RBL and mucosal mast cells were reported to be insensitive to com-
pound 48/80 and sodium cromoglycate, and to degranulate after chal-
lenge with the Ca 2+ ionophore A23187 [30]. In addition, both RBL
and mucosal mast cells were shown to contain granules that can be
stained by toluidine blue and alcian blue, but not safranin, the latter
being capable of staining granules from connective tissue mast cells.
While connective tissue mast cells predominantly contain the rat
mast cell protease-I (RMCP-I), RMCP-II is predominant in both RBL
and mucosal mast cells. Moreover, the histamine content in RBL and
mucosal mast cells was reported to be about 0.1–1% of the amount
found in connective tissue mast cells [29]. Considering that RBL-2H3
cells can be easily cultivated and present considerable homogeneity,
this cell line was used in the present work as a study model for muco-
sal mast cells.
To assess RBL-2H3 cell degranulation we quantified the stimulated
release of β-hexosaminidase, widely accepted as a marker for mast
cell degranulation, since this enzyme is stored in mast cell secretory
granules along with allergic mediators such as histamine, and is re-
leased when mast cells are immunologically stimulated to degranulate
[31,32]. Pretreatment of IgE-sensitized RBL cells with pyridovericin,
followed by cell stimulation with antigen (DNP-BSA), resulted in a
strong and concentration-dependent inhibition of β-hexosaminidase
release (Fig. 2). The calculated IC50 for pyridovericin-mediated inhibi-
tion of degranulation was 7.4± 1.2 μM. It is important to mention that
the inhibitory activity of pyridovericin was dramatically more potent
than that exhibited by the mast cell stabilizer ketotifen fumarate
(Fig. 2, IC50 = 154.7 ±6.4 μM), a commonly used positive control for
mast cell degranulation assays [33,34]. Pyridovericin also exhibited a
quite similar inhibitory activity reported for the flavonoid quercetin
(IC50 = 7.9 ±0.4 μM), a very well-known inhibitor of mast cell degran-
ulation (Fig. 2) [32,35,36].
Incubation of RBL-2H3 cells with pyridovericin for 1 h caused a
minor and not significant decrease in cell viability only at the highest
tested concentration of pyridovericin (100 μM), suggesting that its
Fig. 2. Pyridovericin inhibits FcεRI-mediated mast cell degranulation. IgE-Sensitized
RBL-2H3 cells were treated with pyridovericin (0.5–100 μM) for 20 min, and stimulated
with antigen (Ag, DNP-BSA, 0.4 μg/mL). Following a 1 h incubation period, cell superna-
tant was incubated with 1.2 mM MUG for addition 30 min. Degranulation was quantified
through fluorescence measurements of methyl-umbelliferone, product of MUG-cleavage
mediated by β-hexosaminidase. Ketotifen fumarate and quercetin, both at 100 μM,
were used as positive controls. Data is expressed as mean±SD of 3 independent experi-
ments. One-way ANOVA, followed by Dunnett's test, indicates statistical significance of
**pb 0.01 and ***pb 0.001, in comparison to antigen-control sample. # refers to vehicle
(DMSO) control sample.
 M. de Souza Santos et al. / International Immunopharmacology 15 (2013) 532–538
Fig. 3. Pyridovericin causes a minor decrease in both cell viability and β-hexosaminidase
activity. RBL-2H3 cells were incubated with pyridovericin (1–100 μM) for 1 h. Cell viabil-
ity was assessed by fluorescence measurements of resorufin, resulting from the reduction
of resazurin by viable cells. For β-hexosaminidase activity assay, cell supernatant was
incubated with pyridovericin (1–100 μM) and 1.2 mM MUG solution for 1 h at 37 °C.
Quantification of β-hexosaminidase activity was based on fluorescence measurements
of methyl-umbelliferone. One representative experiment of three, for each assay, is
shown. The error bars represent SD of triplicate samples. One-way ANOVA, followed by
Dunnett's test, indicates statistical significance of *pb 0.05 and **pb 0.01, in comparison
to untreated control samples.
inhibitory activity against degranulation was not caused by a cytotoxic
effect (Fig. 3). In addition, the effect of pyridovericin on the
β-hexosaminidase enzyme activity was also performed to clarify
whether its action was attributed to either the inhibition of the enzyme
or the inhibition of its secretion, the latter being the desired action.
Pyridovericin slightly inhibited β-hexosaminidase activity only at the
higher range of tested concentrations (Fig. 3). These results are consis-
tent with a primary effect of pyridovericin on reducing the release of
β-hexosaminidase, as a result of its inhibitory activity against stimulat-
ed mast cell degranulation.
Interestingly, consistent with pyridovericin being a competitive in-
hibitor, its anti-degranulation activity could be rapidly reversed (Fig. 4).
When RBL-2H3 cells were pretreated with 100 μM pyridovericin,
Fig. 4. Pyridovericin-mediated inhibition of antigen-induced mast cell degranulation is re-
versible. IgE-sensitized mast cells were treated with 100 μM pyridovericin (pyr), 100 μM
quercetin (que) or 300 μM ketotifen fumarate (ket) for 20 min, after which the metabolites
were removed or not by exhaustive washing, followed by antigen stimulation for additional
1 h. Next, cell supernatant was incubated with 1.2 mM MUG for addition 30 min. Degran-
ulation was quantified through fluorescence measurements of methyl-umbelliferone,
product of MUG-cleavage mediated by β-hexosaminidase. Data is expressed as mean±
SD of 3 independent experiments. One-way ANOVA, followed by Dunnett's test, indicates
statistical significance of ***pb 0.001, in comparison to antigen-control sample.
535
Fig. 5. Pyridovericin inhibits FcεRI-dependent and independent degranulation responses
with similar potency. IgE-sensitized RBL-2H3 cells were treated with pyridovericin
(0.5–100 μM) for 20 min, and stimulated with either antigen (Ag, DNP-BSA, 0.4 μg/mL)
or thapsigargin (Tg, 200 nM). Following a 1 h incubation period, cell supernatant was
incubated with 1.2 mM MUG for addition 30 min. Degranulation was quantified through
fluorescence measurements of methyl-umbelliferone, product of MUG-cleavage mediated
by β-hexosaminidase. One representative experiment of three is shown. The error bars
represent SD of triplicate samples. One-way ANOVA, followed by Dunnett's test, indicates
statistical significance of *pb 0.05, **pb 0.01, and ***pb 0.001, in comparison to either
antigen- or thapsigargin-control samples.
followed by antigen stimulation, mast cell degranulation was inhibited
in 70%. However, the removal of pyridovericin of the cell medium, by ex-
haustive washing, prior to antigen addition, completely annulled its in-
hibitory activity. Both quercetin and ketotifen were also shown to
reversibly inhibit antigen-induced mast cell degranulation (Fig. 4).
To understand the mechanisms through which pyridovericin would
exert its inhibitory effect on degranulation, mast cell degranulation was
then stimulated by thapsigargin. Thapsigargin activates SOCE by deple-
tion of ER Ca2+ stores via inhibition of the SERCA (Sarco/Endoplasmic
Reticulum Ca2+) ATPase. Therefore, this drug bypasses the activation
of upstream Ca 2+ mobilization signaling events to cause degranulation.
Thapsigargin-induced degranulation of RBL cells was substantially
inhibited by pyridovericin, in a concentration-dependent manner
(Fig. 5). Moreover, it could be noticed that pyridovericin inhibited
both antigen- and thapsigargin-mediated degranulation responses
with a very similar potency (IC50 values of 7.4± 1.2 and 6.3 ± 0.4 μM,
Fig. 6. Pyridovericin suppresses Ca2+ responses to antigen- and thapsigargin-cell stim-
ulation. RBL mast cells, sensitized with anti-DNP IgE and loaded with Fluo-4, were
stimulated either with antigen (Ag, 0.4 μg/mL) or thapsigargin (Tg, 200 nM). Arrow in-
dicates the moment of stimuli addition. One representative experiment of three is
shown.
536 M. de Souza Santos et al. / International Immunopharmacology 15 (2013) 532–538
Fig. 7. Pyridovericin inhibits antigen-induced secretion of IL-4 from mast cells.
IgE-sensitized RBL-2H3 cells were treated with pyridovericin (0.1–50 μM) and stimu-
lated with antigen (0.4 μg/mL) for 4 h, after which cell supernatant was collected for
quantification of secreted IL-4 by ELISA. For cytotoxicity assay, cells were treated
with pyridovericin at the same concentration range for 4 h. Cell viability was assessed
by fluorescence measurements of resorufin, resulting from the reduction of resazurin
by viable cells. Data is expressed as mean ± SD of 3 independent experiments.
One-way ANOVA, followed by Dunnett's test, indicates statistical significance of
*p b 0.05 and ****p b 0.001, in comparison to antigen-control sample.
for antigen- and thapsigargin-stimulation, respectively). These results
suggest that (a) FcεRI-proximal signaling events, such as phosphoryla-
tion of Src-family kinases may not be targets for pyridovericin inhibition
and (b) the molecular targets of pyridovericin might be shared by both
IgE- and thapsigargin-mediated degranulation pathways.
As the transient increase in intracellular Ca2+ concentration is one of
the earliest events common to both antigen- and thapsigargin-induced
mast cell degranulation [37], Ca2+ signals were analyzed in the absence
of pyridovericin and after a 20-minute treatment of cells with the me-
tabolite. Cell stimulation with both antigen and thapsigargin caused a
robust increase in cytoplasmic Ca2+ levels, whereas cell pre-treatment
with 10 μM pyridovericin strongly suppressed both Ca2+ responses
(Fig. 6). These results are consistent with inhibition of Ca 2+ mobiliza-
tion being one of the leading mechanisms by which pyridovericin im-
pairs mast cell degranulation.
Increase in cytosolic Ca 2+ concentration is also required for the
expression of inflammatory cytokines [38]. In this way, the levels
of secreted interleukine-4 (IL-4), following cell incubation with
pyridovericin and stimulation with antigen, were also quantified. As
shown in Fig. 7, IL-4 secretion was almost completely abolished in
the presence of pyridovericin. Cell viability measurements, assessed
in similar experimental conditions to that used for the IL-4, indicated
no pyridovericin-mediated cytotoxicity.
4. Discussion
As part of an ongoing study to identify novel inhibitors of mast cell
activation, a key event for the development of allergic diseases, we re-
port here that the fungal metabolite pyridovericin strongly sup-
presses both degranulation and secretion of IL-4 from RBL-2H3 cells
stimulated by antigen. Those findings bring new insight to the phar-
macological usability of this metabolite, which was isolated and iden-
tified for the first time over 10 years ago, and, since then, has been
shown to work only as a weak inhibitor of tyrosine kinases from cul-
tured cells [20].
The anti-degranulation activity of pyridovericin was shown to be
comparable to that of quercetin and other structure-related flavonoids
(IC50 at the low micromolar range) (Fig. 2, [32]). It is interesting to
note that most of the extracts and isolated metabolites obtained from
natural sources, which were shown to inhibit antigen-stimulated mast
cell degranulation, were also found to not affect or marginally inhibit
degranulation caused by stimuli other than antigen. For instance, Kim
and coworkers (2009) reported that the flavonoid morin specifically
inhibits antigen-induced mast cell degranulation [4]. Lee and others
(2008) found that the leaf extract of Camellia japonica inhibits
antigen-stimulated degranulation but is inactive against ionomycin-
and thapsigargin-activated mast cells [39]. Quercetin was previously
shown to inhibit histamine release induced by antigen but to have little
effect on ionophore-stimulated release of histamine [40]. The specificity
of many inhibitors for antigen-mediated degranulation is consistent
with their reported primary effect on the activity of FcεRI-proximal en-
zymes, such as the src-family kinases Syk, Lyn, and Fyn [4,13,39,41–43].
Considering that pyridovericin similarly inhibited both antigen- and
thapsigargin-induced degranulation, and was previously shown to
weakly inhibit tyrosine kinases [20], we propose that this metabolite
presents a distinct mechanism of inhibition of mast cell activity in com-
parison to many other natural compounds.
Although the molecular mechanisms of pyridovericin-mediated in-
hibition of mast cell degranulation remain to be determined, we report
here that this metabolite inhibits Ca2+ signaling (Fig. 6), which is a crit-
ical step for degranulation. Mast cell degranulation involves granule
translocation, granule docking and, ultimately, granule fusion with the
plasma membrane. Essential to this process are SNARE [soluble NSF
(N-ethyl-maleimide-sensitive factor) attachment protein receptor]
proteins that lie on the opposing cellular membranes (vesicular and
plasma membrane) to form stable multimeric complex that catalyzes
fusion. Although SNARE proteins by themselves do not bind Ca2+, the
membrane-anchored protein synaptotagmin, binds Ca2+, which is
followed by synaptotagmin oligomerization and combined interaction
with SNAREs and membrane phospholipids to promote membrane fu-
sion and release of granular content [44,45]. Similarly to its inhibitory
action against degranulation, pyridovericin was shown to cause a com-
parable decrease in the cytoplasmic Ca2+ levels of cells stimulated with
either antigen or thapsigargin (Fig. 6). As a control experiment, querce-
tin was shown to strongly inhibit Ca 2+ responses to antigen, but was
considerably less effective in inhibiting Ca 2+ mobilization induced by
thapsigargin (Figs. S12). As for degranulation, the results suggest that
pyridovericin is involved in the inhibition of latter signaling events,
e.g. distal FcεRI signals, required for elevation of intracellular Ca 2+ con-
centration. Mast cells do not exhibit voltage gated Ca 2+ channels and
stimulated release of Ca 2+ from ER stores (by either antigen or
thapsigargin) is sufficient to activate Ca2+ influx (SOCE) necessary for
degranulation [46]. Because the depletion of Ca2+ ER stores occurs by
different mechanisms whether cells are treated with either antigen or
thapsigargin, it is appealing to speculate that the mechanism of action
of pyridovericin could involve inhibition of Ca2+ entry. The Ca2+
entry pathway is mediated by Ca 2+ release-activated Ca2+ channels.
Upon stimulated emptying of ER Ca2+ stores, STIM1, an ER lumen-
resident Ca2+ sensor, oligomerizes and translocates to ER-plasma
membrane junctions where it binds to and activates Orai1, a tetraspan
Ca2+ channel protein, leading to channel-gate opening and massive
Ca2+ entry [7,46,47]. All indications point to STIM1 and Orai1 as being
essential for SOCE activity in mast cells and also to be absolutely re-
quired for FcεRI-mediated mast cell activation both in vitro and in
vivo [47]. For instance, it has been shown that STIM1- or Orai1-
deficient mast cells exhibit impaired Ca2+ influx, which correlated
with dramatically suppressed mast cell degranulation [44,48].
Interestingly, elevation of cytosolic Ca2+ levels is a common signal
for both mast cell degranulation and cytokine production and secretion.
For instance, the cytokine-related transcriptional factor, NFAT, was
shown to become activated via Ca2+/calcinerium-mediated dephos-
phorylation of this enzyme [38,49]. Consistent with an important role
for the Ca 2+/calcinerium/NFAT pathway during the production of cyto-
kines, Andrade and coworkers (2011) reported that similar levels of
tumor necrosis factor-alpha (TNF-α) could be produced when mast
 M. de Souza Santos et al. / International Immunopharmacology 15 (2013) 532–538
cells were stimulated by either antigen or thapsigargin and that secre-
tion was almost completely impaired by addition of the calcinerium
inhibitor cyclosporine A [49]. As for TNF-α, IL-4 production relies on
NFAT-mediated regulation of IL-4 gene promoter [50], which under-
scores a role for Ca2+ during stimulated production of IL-4. In agreement
with those findings, we showed that pyridovericin was responsible for a
concentration-dependent suppression of antigen-stimulated secretion
of IL-4 (Fig. 7). IL-4 is a pleiotropic cytokine which exerts both indirect
and direct effects on mast cell responses [50,51]. IL-4 production, during
antigen presentation, directs näive T-cell development to Th2-subset.
Th2 cells secrete IL-4, eliciting IgE production from activated B cells,
which allows for mast cell activation through antigen-IgE-FcεRI path-
way. Stimulated mast cells produce more IL-4, propagating the Th2 re-
sponse [51]. Another potentially important activity of IL-4 is its ability
to induce the expression of vascular cell adhesion molecule (VCAM)-1
on the endothelial cells [52,53]. VCAM-1 plays an important role for in-
creased adhesiveness of the endothelium for T cells, eosinophils, baso-
phils, and monocytes, whose elevated levels have been associated with
allergic diseases such as asthma [52–55]. Yamaguchi and coworkers
reported that IL-4 can induce expression of FcεRI on the mast cell surface
and increase secretion of allergic mediators from mast cells stimulated
with anti-IgE [56]. IL-4 was additionally shown to control mast cell
homeostasis, presenting inhibitory effects on mast cell function and
survival [51].
In summary, we report here the initial studies that identify
pyridovericin, a secondary metabolite isolated from the
entomopathogenic fungi B. bassiana, as a promising leading compound
for the development of novel anti-allergic drugs. Its pharmacological
activity was attributed to its inhibitory activity against the secretion of
allergic mediators from RBL-2H3 cells, a model system for studies of
FcεRI-regulated secretion in mast cells. Pyridovericin was shown to be
a competitive inhibitor of antigen-induced degranulation and to impair
stimulated IL-4 secretion, both of which were correlated to the
pyridovericin-mediated suppression of Ca 2+ influx. Further studies to
dissect the molecular mechanisms responsible for the inhibitory activity
of pyridovericin comprise one of the next steps that could corroborate
the therapeutical suitability of this metabolite. It is also of interest to
demonstrate that pyridovericin is active in animal models. It is notewor-
thy that the physicochemical properties of pyridovericin are in good
agreement with Lipinski's rule of five [57], which advocates, at least in
theory, for the drug-like properties of this metabolite.
Acknowledgments
The authors thank Laila Aparecida Deliberto for technical support.
This work was supported by Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES), grant # 1277/10-3, Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant # 2008/
01712-6 and Instituto Nacional de Ciência e Tecnologia de Bioanalítica.
The authors declare they have no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.intimp.2013.01.017.
References
[1] Douwes J, Brooks C, van Dalen C, Pearce N. Importance of allergy in asthma: an
epidemiologic perspective. Curr Allergy Asthma Rep 2011;11:434–44.
[2] Small P, Kim H. Allergic rhinitis. Allergy Asthma Clin Immunol 2011;7:S1–3.
[3] Kupczyk M, Haahtela T, Cruz AA, Kuna P. Reduction of asthma burden is possible
through National Asthma Plans. Allergy 2010;65:415–9.
[4] Kim JW, Lee JH, Hwang BY, Mun SH, Ko NY, Kim do K, et al. Morin inhibits Fyn
kinase in mast cells and IgE-mediated type I hypersensitivity response in vivo.
Biochem Pharmacol 2009;77:1506–12.
[5] Masuda ES, Schimitz J. Syk inhibitors as treatment for allergic rhinitis. Pulm
Pharmacol Ther 2008;21:461–7.
537
[6] Berridge MJ, Dawson RM, Downes CP, Heslpo JP, Irvine RF. Changes in the levels of ino-
sitol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.
Biochem J 1983;212:473–82.
[7] Vasuvedan L, Jeromin A, Volpicelli-Daley L, De Camilli P, Holowka D, Baird B. The
β- and γ-isoforms of type I PIP5K regulate distinct stages of Ca2+ signaling in
mast cells. J Cell Sci 2009;122:2567–74.
[8] Stevens RL, Austen KF. Recent advances in the cellular and molecular biology of
mast cells. Immunol Today 1989;10:381–6.
[9] Moriya K, Rivera J, Odom S, Sakuma Y, Muramoto K, Yoshiuchi T, et al. ER-27319,
an acridone-related compound, inhibits release of antigen-induced allergic medi-
ators from mast cell by selective inhibition of fcepsilon receptor I-mediated acti-
vation of Syk. Proc Natl Acad Sci USA 1997;94:12539–44.
[10] Rossi AB, Herlaar E, Braselmann S, Huynh S, Taylor V, Frances R, et al. Identifica-
tion of the Syk kinase inhibitor R112 by a human mast cell screen. J Allergy Clin
Immunol 2006;118:749–55.
[11] Kambayashi T, Koretzky GA. Proximal signaling events in Fc epsilon RI-mediated
mast cell activation. J Allergy Clin Immunol 2007;119:544–52.
[12] Moon PD, Lee BH, Jeong HJ, An HJ, Park SJ, Kim HR, et al. Use of scopoletin to in-
hibit the production of inflammatory cytokines through inhibition of the
IkappaB/NF-kappaB signal cascade in the human mast cell line HMC-1. Eur J
Pharmacol 2007;555:218–25.
[13] Nakano N, Nishiyama C, Tokura T, Nagasako-Akazome Y, Ohtake Y, Okumura K,
et al. Procyanidin C1 from apple extracts inhibits Fc epsilon RI-mediated mast
cell activation. Int Arch Allergy Immunol 2008;147:213–21.
[14] Tanufuji S, Aizu-Yokota E, Funakoshi-Tago M, Sonoda Y, Inoue H, Kasahara T.
Licochalcones suppress degranulation by decreasing the intracellular Ca2+ level
and tyrosine phosphorylation of ERK in RBL-2H3 cells. Int Immunol 2010;10:
769–76.
[15] Mishra BB, Tiwari VK. Natural products: an evolving role in future drug discovery.
Eur J Med Chem 2011;46:4769–807.
[16] Newman DJ, Cragg GM. Natural products as sources of new drugs over the
30 years from 1981 to 2010. J Nat Prod 2012;75:311–35.
[17] Hoffmeister D, Keller NP. Natural products of filamentous fungi: enzymes, genes,
and their regulation. Nat Prod Rep 2007;24:393–416.
[18] Keller NP, Turner G, Bennett JW. Fungal secondary metabolism — from biochem-
istry to genomics. Nat Rev Microbiol 2005;3:937–47.
[19] Takahashi S, Uchida K, Kaninuma N, Hashimoto R, Yanagisawa T, Nakagawa A. The
structures of pyridovericin and pyridomacrolidin, new metabolites from the
entomopathogenic fungus, Beauveria bassiana. J Antibiot 1998;51:1051–4.
[20] Takahashi S, Kakinuma N, Uchida K, Hashimoto R, Yanagisawa T, Nakagawa A.
Pyridovericin and pyridomacrolidin: novel metabolites from entomopathogenic
fungi, Beauveria bassiana. J Antibiot 1998;51:596–7.
[21] Cheng Y, Scheneifer B, Riese U, Schubert B, Li Z, Hamburger M. (+)-N-
Deoxymilitarinone A, a neuritogenic pyridone alkaloid from the insect pathogenic
fungus Paecilomyces farinosus. J Nat Prod 2006;69:436–8.
[22] de Silva ED, Geiermann AS, Mitova MI, Kuegler P, Blunt JW, Cole AL, et al. Isolation
of 2-pyridone alkaloids from a New Zealand marine-derived penicillium species.
J Nat Prod 2009;72:477–9.
[23] Jessen HJ, Gademann K. 4-Hydroxy-2-pyridone alkaloids: structures and synthetic
approaches. Nat Prod Rep 2010;27:1168–85.
[24] Khanna P, Sundari SS, Kumar NJ. Production, isolation and partial purification of
xylanase from Aspergillus sp. World J Microbiol Biotechnol 1995;11:242–3.
[25] Barsumian EL, Isersky C, Petrino MG, Siraganian RP. IgE-induced histamine release
from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing
clones. Eur J Immunol 1981;11:317–23.
[26] Naal RMZG, Tabb J, Holowka D, Baird B. In situ measurement of degranulation as a
biosensor based on RBL-2H3 masr cells. Biosens Bioelectron 2004;20:791–6.
[27] Posner RG, Lee B, Conrad DH, Holowka D, Baird B, Goldstein B. Aggregation of
IgE-receptor complexes on rat basophilic leukemia cell does not change the intrinsic
affinity but can alter the kinetics of the ligand–IgE interaction. Biochemistry
1992;31:5350–6.
[28] Hardy R. In: Weir DM, editor. Handbook of experimental immunology. Oxford:
Blackwell Scientific Publications; 1986.
[29] Seldin DC, Adelman S, Austen KF, Hein A, Caufield JP, Woodbury RG. Homology of
the rat basophilic leukemia cell and the rat mucosal mast cell. Proc Natl Acad Sci
USA 1985;82:3871–5.
[30] Passante E, Frankish N. The RBL-2H3 cell line: its provenance and suitability as a
model for mast cell. Inflamm Res 2009;58:737–45.
[31] Lee E, Choi EJ, Cheong H, Kim YR, Ryu SY, Kim KM. Anti-allergic actions of the
leaves of Castanea crenata and isolation of an active component responsible for
the inhibition of mast cell degranulation. Arch Pharm Res 1999;22:320–3.
[32] Matsuda H, Morikawa T, Ueda K, Managi H, Yoshikawa M. Structural require-
ments of flavonoids for inhibition of antigen-induced degranulation, TNF-alpha
and IL-4 production from RBL-2H3 cells. Bioorg Med Chem 2002;10:3123–8.
[33] Serna H, Porras M, Vergara P. Mast cell stabilizer ketotifen [4-(1-methyl-4-
piperidylidene)-4h-benzo[4,5]cyclohepta[1,2-b]thiophen-10(9H)-one fumarate]
prevents mucosal mast cell hyperplasia and intestinal dysmotility in experimen-
tal Trichinella spiralis inflammation in the rat. J Pharmacol Exp Ther 2006;319:
1104–11.
[34] Lambiase A, Micera A, Bonini S. Multiple action agents and the eye: do they really
stabilize mast cells? Curr Opin Allergy Clin Immunol 2009;9:454–65.
[35] Middleton Jr E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on
mammalian cells: implications for inflammation, heart disease, and cancer.
Pharmacol Rev 2000;52:673–751.
[36] Chirumbolo S. The role of quercetin, flavonols and flavones in modulating inflam-
matory cell function. Inflamm Allergy Drug Targets 2010;9:263–85.
538 M. de Souza Santos et al. / International Immunopharmacology 15 (2013) 532–538
[37] Ansdrásfalvy M, Péterfy H, Tóth G, Matkó J, Abramson J, Kerekes K, et al. The beta
subunit of the type I Fcepsilon receptor is a target for peptides inhibiting
IgE-mediated secretory response of mast cells. J Immunol 2005;175:2801–6.
[38] Holowka D, Calloway N, Cohen R, Gadi D, Lee J, Smith NL, et al. Roles for Ca2+ mo-
bilization and its regulation in mast cell functions. Front Immunol 2012;3:104.
[39] Lee J-H, Kim J-W, Ko N-Y, Mun S-H, Kim D-K, Kim J-D, et al. Camellia japonica
suppresses immunoglobulin E-mediated allergic response by the inhibition of
Syk kinase activation in mast cells. Clin Exp Allergy 2008;38:794–804.
[40] Fewtrell CM, Gomperts BD. Quercetin: a novel inhibitor of Ca2+ influx and exocy-
tosis in rat peritoneal mast cells. Biochim Biophys Acta 1977;469:52–60.
[41] Itoh T, Ohguchi K, Iinuma M, Nozawa Y, Akao Y. Inhibitory effects of polymethoxy
flavones isolated from Citrus reticulate on degranulation in rat basophilic leuke-
mia RBL-2H3 cells: enhanced inhibition by their combination. Bioorg Med Chem
2008;16:7592–8.
[42] Lee JH, Kim JW, Ko NY, Mun SH, Her E, Kim BK, et al. Curcumin, a constituent of
curry, suppresses IgE-mediated allergic response and mast cell activation at the
level of Syk. J Allergy Clin Immunol 2008;121:1225–12231.
[43] Lee JH, Kim JW, Ko NY, Mun SH, Kim DK, Kim JD, et al. Mast cell-mediated allergic
response is suppressed by Sophorae flos: inhibition of Src-family kinases. Exp Biol
Med 2008;233:1271–9.
[44] Baba Y, Nishida K, Fujii Y, Hirano T, Kurosaki T. Essential function for the calcium
sensor STIM1 in mast cell activation and anaphylactic responses. Nat Immunol
2008;9:81–8.
[45] Blank U, Rivera J. The ins and outs of IgE-independent mast-cell exocytosis.
Trends Immunol 2004;25:266–73.
[46] Di Capite J, Parekh AB. CRAC channels and Ca2+ signaling in mast cells. Immunol
Rev 2009;23:45–58.
[47] Ma HT, Beaven MA. Regulation of Ca2+ signaling with particular focus on mast
cells. Crit Rev Immunol 2009;29:155–86.
[48] Vig M, DeHaven WI, Bird GS, Billingsley JM, Wang H, Rao PE, et al. Defective mast
cell effector functions in mice lacking the CRACM1 pore subunit of store-operated
calcium release-activated calcium channels. Nat Immunol 2008;9:89–96.
[49] Andrade MV, Iwaki S, Ropert C, Gazinelli RT, Cunha-Melo JR, Beaven MA. Amplifica-
tion of cytokine production through synergistic activation of NFAT and AP-1 follow-
ing stimulation of mast cell with antigen and IL-33. Eur J Immunol 2011;41:760–72.
[50] Frossi B, Rivera J, Hirsch E, Purcillo C. Selective activation of Fyn/PI3K and p38
MAPK regulates IL-4 production in BMMC under nontoxic stress condition.
J Immunol 2007;178:2549–55.
[51] Ryan JJ, Kashyap M, Bailey D, Kennedy S, Speiran K, Brenzovich J, et al. Mast cell ho-
meostasis: a fundamental aspect of allergic disease. Crit Rev Immunol 2007;27:
15–32.
[52] Lampinen M, Carlson M, Håkansson LD, Venge P. Cytokine-regulated accumula-
tion of eosinophils in inflammatory disease. Allergy 2004;59:793–805.
[53] Renauld J-C. New insights into the roles of cytokines in asthma. J Clin Pathol
2001;54:577–89.
[54] Hart PH. Regulation of the inflammatory response in asthma by mast cell prod-
ucts. Immunol Cell Biol 2001;79:149–53.
[55] Yamashita M, Nakayama T. Progress in allergy signal research on mast cells: reg-
ulation of allergic airway inflammation through toll-like receptor 4-mediated
modification of mast cell function. J Pharmacol Sci 2008;106:332–5.
[56] Yamaguchi M, Sayama K, Yano K, Lantz CS, Noben-Trauth N, Ra C, et al. IgE
enhances Fcε receptor I expression and IgE-dependent release of histamine and
lipid mediators from human umbilical cord blood-derived mast cells: synergistic
effect of IL-4 and IgE on human mast cell Fcε receptor I expression and mediator
release. J Immunol 1999;162:5455–65.
[57] Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and devel-
opmental settings. Adv Drug Deliv Rev 2011;46:3–26.