Activation of the NLRP3 Inflammasome by Hyaboron, a New Asymmetric Boron-Containing Macrodiolide from the Myxobacterium Hyalangium minutum

Article abstract of DOI:10.1021/acschembio.8b00659

A Natural Compound Library containing myxobacterial secondary metabolites was screened in murine macrophages for novel activators of IL-1β maturation and secretion. The most potent of three hits in total was a so far undescribed metabolite, which was identified from the myxobacterium Hyalangium minutum strain Hym3. While the planar structure of 1 was elucidated by high resolution mass spectrometry and NMR data yielding an asymmetric boron containing a macrodiolide core structure, its relative stereochemistry of all 20 stereocenters of the 42-membered ring was assigned by rotating frame Overhause effect spectroscopy correlations, ¹H,¹H, and ¹H,¹³C coupling constants, and by comparison of ¹³C chemical shifts to those of the structurally related metabolites tartrolon B-D. The absolute stereochemistry was subsequently assigned by Mosher's and Marfey's methods. Further functional studies revealed that hyaboron and other boronated natural compounds resulted in NLRP3 inflammasome dependent IL-1β maturation, which is most likely due to their ability to act as potassium ionophores. Moreover, besides its inflammasome-stimulatory activity in human and mouse cells, hyaboron (1) showed additional diverse biological activities, including antibacterial and antiparasitic effects.

Full text of DOI:10.1021/acschembio.8b00659

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Article
Activation of the NLRP3 Inflammasome by Hyaboron, a new Asymmetric
Boron-Containing Macrodiolide from the Myxobacterium Hyalangium minutum
Frank Surup, Dhruv Chauhan, Jutta Niggemann, Eva Bartok, Jennifer Herrmann,
Matthias Keck, Wiebke Zander, Marc Stadler, Veit Hornung, and Rolf Müller
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00659 • Publication Date (Web): 05 Sep 2018
Downloaded from http://pubs.acs.org on September 5, 2018
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Activation of the NLRP3 Inflammasome by Hyaboron, a new
Asymmetric Boron-Containing Macrodiolide from the Myxobacte-
rium Hyalangium minutum
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Frank Surup,‡,⊥ ,+ Dhruv Chauhan,†,+ Jutta Niggemann,‡ Eva Bartok,§ Jennifer Herrmann,∥,⊥ Matthias
‡,
‡,
‡,
Keck, Wiebke Zander, Marc Stadler, ⊥ Veit Hornung,*,†,# and Rolf Müller*,‡, ,
∥ ⊥
∇
○
‡Helmholtz Center for Infection Research (HZI) Department Microbial Drugs Inhoffenstraße 7, 38124 Braunschweig, Gerꢀ
many
†Gene Center and Department of Biochemistry, LudwigꢀMaximiliansꢀUniversität München, Munich, FeodorꢀLynenꢀStraße
25, Munich, 81377, Germany
§Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, SigmundꢀFreudꢀStraße 25, University of
Bonn, 53127 Bonn, Germany
∥Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research and Department
of Pharmacy, Saarland University Campus, Building E8.1, 66123 Saarbrücken, Germany
⊥German Centre for Infection Research Association (DZIF), partner site HannoverꢀBraunschweig, Inhoffenstraße 7, 38124
Braunschweig, Germany
#Center for Integrated Protein Science (CIPSM), LudwigꢀMaximiliansꢀUniversität München, Munich, FeodorꢀLynenꢀStraße
25, Munich, 81377, Germany
ABSTRACT: A Natural Compound Library containing myxobacterial secondary metabolites was screened in murine macrophages
for novel activators of ILꢀ1β maturation and secretion. The most potent of three hits in total was a so far undescribed metabolite,
which was identified from the myxobacterium Hyalangium minutum strain Hym3. While the planar structure of 1 was elucidated by
HRMS and NMR data yielding an asymmetric boron containing macrodiolide core structure, its relative stereochemistry of all 20
stereo centers of the 42ꢀmembered ring was assigned by ROESY correlations, 1H,1H and 1H,13C coupling constants, and by comparꢀ
ison of 13C chemical shifts to those of the structurally related metabolites tartrolon BꢀD. The absolute stereochemistry was subseꢀ
quently assigned by Mosher’s and Marfey’s methods. Further functional studies revealed that hyaboron and other boronated natural
compounds resulted in NLRP3 inflammasome dependent ILꢀ1β maturation, which is most likely due to their ability to act as potasꢀ
sium ionophores. Moreover, besides its inflammasomeꢀstimulatory activity in human and mouse cells, hyaboron (1) showed addiꢀ
tional diverse biological activities, including antibacterial and antiparasitic effects.
The human immune system continuously encounters various
pathogens. These threats are detected at the molecular level
via the soꢀcalled pathogen recognition receptors (PRR) of the
innate immune system, which sense the distinct pathogen
associated molecular patterns (PAMP) of invading microorꢀ
ganisms. The distinction between self and nonꢀself molecules
is critical to pathogen recognition and clearance. Nonetheless,
the immune system is not always capable of eradicating pathꢀ
ogenic microorganisms on its own.1 Antibiotics are a vital
therapeutic approach to the treatment of many bacterial infecꢀ
tions. These compounds specifically act against bacterial proꢀ
cesses and targets such as cell wall synthesis or transcriptional
and translational machineries.2 However, the extensive use of
broadꢀspectrum antibiotics in past years has resulted in the
emergence of multiꢀdrug resistant pathogens.3 Therefore, the
struggle against bacterial pathogens urgently calls for novel
strategies such as narrowꢀspectrum antibiotics, which are
effective against selective species of one bacterial class, as
well as the use of adjuvant immunotherapies for the activation
of the host immune system.4 Since broad immune activation
could be detrimental to the host, targeted immunotherapies are
needed, yet such approaches require detailed knowledge of
which pathways control protective immune responses and
which are responsible for inappropriate tissue damage at the
site of infection.5
The inflammasomeꢀdependent interleukinꢀ1β (ILꢀ1β) pathꢀ
way plays an important role in the control and elimination of
bacterial pathogens.6 Inflammasomes are cytosolic, macromoꢀ
lecular complexes which are formed upon sensing PAMPs or
danger associated molecular patterns (DAMPs).7 Inflamꢀ
masome complexes typically consist of a sensor protein such
as a NODꢀlike receptor (e.g. NLRP3), the adapter molecule
ASC (apoptosis associated speck like protein containing
caspase activation and recruitment domain) and the protease
caspaseꢀ1. Upon activation, the sensor protein acts as a seed to
initiate the prionꢀlike formation of large filamentous ASC
structures. In turn, these ASC filamentous structures recruit
and activate caspaseꢀ1, which then catalyzes the proteolytic
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Figure 1. (A) Natural Compound Library of the DZIF was screened (1 µM) using J774 macrophage cell line stably transduced with
iGLuc and constitutively expressing NLRP3. The iGLuc signal was measured in supernatant after 2 hr. The figure is representative of
two independent experiments performed in duplicate. Error bars represent mean + SEM. AU, arbitrary units. (B) Calculations of Zꢀfactor
of iGLuc screening.
strain Hym3. The quasimolecular HRESIMS ion peak at m/z
1068.5710 provided the molecular formula C56H82BNO18. The
presence of boron was deduced from a characteristic [Mꢀ1]
peak with 25% intensity in the molecular ion cluster.
maturation of interleukinꢀ1β (ILꢀ1β) and its family member
ILꢀ18.7 In general, activation of inflammasomes is a twoꢀstep
process where signal 1 acts as the priming signal to induce the
expression of proꢀILꢀ1β and signal 2 is required to activate
inflammasome sensor itself. Additionally, for the NLRP3
inflammasome, signal 1 facilitates priming of NLRP3 expresꢀ
sion as well as its activation at transcriptional and postꢀ
transcriptional level.8,9
After isolation of 15.6 mg of hyaboron (1) from a large
1
scale fermentation, proton and H,13C HSQC spectra revealed
the existence of seven methyl, 14 methylene, nine olefinꢀ
ic/aromatic methine (two with dual intensity), and 15 aliphatic
methine groups, leaving six exchangeable protons. The carbon
spectrum resolved 11 additional carbon atoms: three carbonꢀ
yls, one sp2 and four oxygenated sp3 hybridized quaternary
To identify novel immune adjuvants for antiꢀinfective therꢀ
apies or targeted modulators of the host immune system we
screened the Natural Compound Library (NCL) of the German
Centre for Infection Research (DZIF), a collection of 259
purified small molecules (compound number at the time point
of screening, 2013/2014) derived from myxobacteria and other
natural sources for their ability to induce ILꢀ1β secretion
(Figure 1A). The library was provided in a readyꢀtoꢀscreen
format by the Thematic Infrastructure (TI) NCL, which is now
part of the Thematic Translational Unit (TTU) Novel Antibiotꢀ
ics of DZIF.
1
carbons. H,1H COSY and TOCSY correlations showed five
spin systems (Figure S1, Table S1). The largest ones were
1
connected by H,13C HMBC correlations (Figure S1) to form
the 42ꢀmembered core structure characteristic for tartrolonꢀ
type metabolites. An additional sixꢀmembered ring structure
was deduced by the HMBC correlation between 9‒H and C‒
13. A protonated Nꢀmethylphenylalanyl side chain, forming an
inner ion pair with the boronꢀcore structure, was assigned and
connected to C‒11 due to a HMBC correlation from 11‒H to
C‒1”. The C‒14/C‒15 epoxide was indicated by the high field
To this end, we utilized J774 immortalized murine macroꢀ
phages that were stably transduced with iGLuc, a Gaussia
luciferaseꢀbased proteolytic reporter for inflammasome activiꢀ
ty.10 The iGLuc reporter allowed the measurement of Gaussia
luciferase activity as a readout for proteolytic ILꢀ1β maturaꢀ
tion.10,11 Additionally, we stably transduced the cell line with
NLRP3 under the constitutive EF1α promoter, which elimiꢀ
nated the need for NFꢀkB priming of NLRP3 expression prior
to compound screening.8,12 In our screening system, stimulaꢀ
tion with the NLRP3 activator nigericin yielded a Zꢀfactor of
0.84 (Figure 1B).13ꢀ15 The hit threshold for activation was then
defined as luciferase activity more than three standard deviaꢀ
tions from the mean of all wells. Two independent screenings
resulted in the reproducible identification of three activators:
vioprolide A,16 tartrolon B17 and hyaboron (1, Figure 1A),
corresponding to 1.1% of compounds screened. Interestingly,
hyaboron (1), the strongest hit, was previously isolated but not
structurally elucidated in our laboratory. Consequently, we
decided to perform a comprehensive structural examination,
and 1 was isolated from the extracts of Hyalangium minutum,
shifts of oxymethines 14‒H (δH 12.81) and 15‒H (δH 3.10), and
1
further authenticated by large JH14C14 and JH15C15 values of
177 and 174 Hz, respectively. Vicinal coupling constants of
11.4 Hz indicated Z configurations of the ꢀ16,17 and ꢀ16’,17’
double bonds, while the coupling constant of 14.2 Hz suggestꢀ
ed the E configuration of the ꢀ14’,15’ double bond.
The relative configuration of 1 was derived from ROESY
correlations, analysis of coupling constants and comparison of
carbon chemical shifts to those of tartrolon B and C, for which
detailed NMR analyses and crystal structures are available.16,17
Particularly, the sixꢀmembered ketal rings had the alkyl subꢀ
stituents at C‒3, C‒4, C‒7 and C‒3’, C‒4’, C‒7’, respectively,
in all equatorial positions according to diaxial ROESY correlaꢀ
tions (Figure S2). ROESY correlations between 4‒OH and 2‒
H & 20’‒H, respectively 2’‒H and 4’‒H & 20‒H indicated an
2R,2’R,4S,4’R,20S,20’S stereochemistry. The sterical proximiꢀ
ty of these protons is indicated in crystal structures of tartolons
B and C.17,18 The analogous assignment was confirmed by
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nearly identical chemical shifts and signal multiplicities in the
related secondary metabolites also includes boromycin from
Streptomyces antibioticus,24 aplasmomycin from a marine S.
griseus,25 tartrolon C18 (from Streptomyces sp.) and borophyꢀ
cin26 (from cyanobacterium Nostoc linckia). Of these macrodiꢀ
olides, the only asymmetric representative discovered to date
is boromycin.
NMR data for these parts in 1. The relative stereochemistry of
the C‒7/C‒8/C‒9 and C‒7’/C‒8’/C‒9’/C‒11’ stereoclusters
was addressed by a detailed Jꢀbased configurational analysis
1
(Figure S3).19 To determine necessary H,1H coupling conꢀ
stants in spite of grave overlap of signals in the 1H NMR specꢀ
trum, a series of 1D TOCSY NMR experiments irradiating at
23‒H3, 9‒H, 11‒H, 21‒H3, 23’‒H3, 9’‒H and 11’‒H was
conducted. Strong ROESY correlations between 9‒H, 13‒OH
and methyl 10”‒H3 indicted their axial positions defining a
9S,11R,13S configuration. Finally, the relative configuration
between the trans epoxide (JH14,H15 = 2.8 Hz) and C‒13 was
By HPLCꢀESIMS analysis, hyaboron (1) could also be deꢀ
tected in crude extracts of Hyalangium minutum, strain
NOCBꢀ2T, the producer of hyaladione, hyafurones, hyꢀ
apyrrolines, and hyapyrones.27,28 This finding might suggest a
broad distribution of hyaboron occurrence in the genus Hyaꢀ
langium minutum.
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established. The large value JH14C13 = 6.6 Hz determined by a
Having elucidated the full structure of hyaboron (1), we folꢀ
lowed up on the primary screen that was conducted in murine
macrophages and investigated the ability of hyaboron and
tartrolon B to activate ILꢀ1β in human peripheral blood monoꢀ
nuclear cells (PBMCs). In this set of experiments, we also
included boromycin and tartrolon A (without a central boron)
alongside the positive control nigericin.14 Nigericin is a wellꢀ
characterized antibiotic from Streptomyces hygroscopicus that
acts as a potassium ionophore. By triggering potassium efflux
from cells along the chemical gradient, it acts as a potent actiꢀ
vator of the NLRP3 inflammasome.29 Similar to nigericin,
hyaboron, tartrolon B and boromycin induced a considerable
ILꢀ1β release into the supernatant of LPSꢀprimed PBMCs
(Figure 3A and B). Tartrolon A, however, did not induce ILꢀ
1β release. At their maximal effective concentrations, tartrolon
B and hyaboron induced approximately 50% of the ILꢀ1β
response that was elicited by nigericin, whereas the activity of
boromycin was approximately 20% of nigericin. Determining
the potency of these compounds by calculating their pEC50
(negative logarithm of half maximum effective concentration)
revealed that tartrolon B had a pEC50 of 6.14±0.15, hyaboron
of 7.47±0.09 and boromycin of 7.27±0.12, whereas the posiꢀ
tive control nigericin had a pEC50 of 8.7±0.02 (Figure 3C).
Jꢀresolved HMBC experiment indicated a gauge configuration
of 14‒H to both oxygens of C‒13. In conjunction with the
observed ROESY correlations of 14‒H to 12‒Hb and 15‒H to
13‒OH and 12‒Ha (Figure S2c), this can only be explained by
an eclipsed configuration of C‒12/C‒14, and a 13R,14R,15R
configuration.
16'
21'
14'
OH OH
23'
22 O
O
O
9'
11'
HO
1
4
7'
4'
O
B–
O
O
7
O
O
O
1'
OH
O
22'
21
O
O
9
23
14
16
1''
9'' O
O
H
N
Similar to nigericin, boromycin has been shown to exert anꢀ
timicrobial activity through its function as a potassium ionoꢀ
phore.30 Moreover, tartrolons have also been shown to trigger
potassium efflux.31 As potassium efflux is required upstream
of the activation of classical NLRP3 inflammasome activaꢀ
tion,29 we hypothesized that all of the boronated compounds
investigated here triggered ILꢀ1β release via activation of the
NLRP3 inflammasome. To address this question, we tested the
activity of these molecules in the presence of increasing conꢀ
centrations of extracellular K+, which has been shown to speꢀ
cifically perturb NLRP3 inflammasome activation by preventꢀ
ing K+ efflux. In addition, we also included the NLRP3ꢀ
specific small molecule inhibitor MCC950 in these studies.32
In order to include dsDNA, as a NLRP3ꢀindependent inflamꢀ
masome stimulus, we conducted these experiments in murine
bone marrow derived macrophages (BMDMs).33 As expected,
increasing the extracellular K+ concentration as well as
MCC950 treatment abrogated ILꢀ1β release by nigericin (Figꢀ
ure 3D), whereas activation of the AIM2 (absent in melanoma
2) inflammasome using double stranded DNA was not affectꢀ
ed by these treatments. Similarly, increasing extracellular K+
concentrations or including MCC950 inhibited ILꢀ1β secretion
by all boronated macrodiolides. In line with the results obꢀ
tained in PBMCs, Tartrolon A did not result in maturation of
+H
5''
3''
1
10''
Figure 2. Structure of hyaboron (1), a novel asymmetric boronꢀ
containing macrodiolide.
Assignment of the absolute stereochemistry of the 9’,11’ꢀ
diol moiety was carried out by Mosher’s method. The ꢀδH
pattern of the MTPA derivatives (Figure S4) with positive
values of 7‒H, 8‒H, 23‒H3 and negative ones of 12‒H2 to 15‒
‒H clearly supports 9’S,11’S configuration.20 Because of the
dimeric biosynthesis of tartrolonꢀtype metabolites,21 this also
defined the stereochemistry of the second hemisphere and
finally showed the same absolute configuration as for tartrolon
B, which was established by Xꢀray data.22 The absolute conꢀ
figuration of the Nꢀmethylꢀphenylalanine side chain was deꢀ
termined by Marfey’s method after alkaline hydrolysis of 1.23
Comparison with authentic standards by HPLCꢀMS analysis
finally revealed S configuration of C2’’ in 1.
Hyaboron shares its boron binding core structure with a few
other boronꢀcontaining macrodiolides (see Figure S5). Its
closest relative, tartrolon B, was isolated from myxobacterium
Sorangium cellulosum.17,22 This small group of structurally
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Figure 3. (AꢀC) Donor PBMCs were primed with LPS for 4 h and stimulated 2 h at the concentrations indicated with the organoborons
hyaboron, tartrolon B, and boromycin (A) as well Tartrolon A, which lacks a central boron, and the canonical NLRP3 stimulus nigeriꢀ
cin (B). ILꢀ1β was measured in cellular supernatants via ELISA. The graphs display maximal ILꢀ1β release (left) and dose titration as a
percentage of the maximum (right) for each compound. For pEC50 determination (C) curve fitting was performed via 4 parameter
logistic nonꢀlinear regression. Data are mean + SEM (n= 4 donors). (D) Effect of different concentrations of extracellular K+ on ILꢀ1β
release. Murine BMDMs were primed with LPS for 4 h and stimulated with nigericin (6.5 µM), dsDNA (1 µg/ml), 20 µM of tartrolon
A, B, hyaboron and boromycin in presence of either different concentrations of extracellular K+ or MCC950 (10 µM). After 8 h, ILꢀ1β
was measured in cellular supernatants via ELISA. Data are mean + SEM (n= 3) (E, F) Immunoblotting of cleaved ILꢀ1β and cleaved
caspaseꢀ1 in BMDMs from WT and mice deficient in Nlrp3. Cells were primed for LPS for 4 h then stimulated with tartrolon A and B
(E) or hyaboron and boromycin (F) as well as the control stimuli dsDNA and nigericin. Cellular lysates (Lys) and supernatants (sup)
were then subjected to SDSꢀPAGE analysis and probed with the indicated antibodies. These data are representative of two independent
experiments.
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pO2: 40 %, V(medium): 5,0 L (5,0 %) for 96 hours in POL/X
ILꢀ1β in murine BMDMs. Furthermore, investigating caspaseꢀ
1 and ILꢀ1β maturation by immunoblotting confirmed the
results obtained by ELISA. All boronated compounds resulted
in the maturation of caspaseꢀ1 and ILꢀ1β in wildtype macroꢀ
phages, similar to the positive controls nigericin and dsDNA
(Fig. 3E and F, left panels). Moreover, comparing wildtype
and Nlrp3ꢀdeficient macrophages validated the specific inꢀ
volvement of the NLRP3 inflammasome in the recognition of
the boronated compounds. To this end, ILꢀ1β and caspaseꢀ1
maturation were completely blunted in Nlrp3ꢀdeficient macroꢀ
phages stimulated with nigericin, tartrolon B, hyaboron or
boromycin, whereas AIM2ꢀdependent inflammasome activaꢀ
tion was fully intact in these cells (Fig. 3E and F, right panels).
Medium (Probion 3 g/L, soluble starch 3 g/L, MgSO4 × 7 H2O
2 g/L, CaCl2 × 2 H2O 0.5 g/L) behenyl alcohol 150 mg/kg
0.15 g/L, vitamin B12 (Cyanocobalamin) 500 µg/L).
Extraction and Isolation:
The XAD resin was eluted with methanol (3 L) and acetone
(3 L). Evaporation of the solvents gave an oily extract (17.3
g). The residue was dissolved in ethyl acetate and water and
the aqueous layer extracted three times with ethyl acetate. The
combined organic layers were evaporated, dried with Na2SO4
and evaporated to dryness to yield 7.5 g crude extract. The
residue was partitioned between methanol containing 5%
water and nꢀheptane. The methanol layer was extracted twice
with nꢀheptane to give 2.0 g of a crude methanol extract. Silica
gel chromatography (column 120 x 50 mm, Silica gel 60 enꢀ
riched with 0.1% Ca, Fluka) using 4% methanol in dichloroꢀ
methane as solvent gave 0.8 g of an enriched product fraction.
Subsequent LHꢀ20 chromatography (4.5 x 85 cm) using diꢀ
chloromethaneꢀmethanol 50:50 as eluent at a flow rate of 2.5
mL/min gave 59.6 mg crude product which was further puriꢀ
fied in two batches by preparative HPLC on VP250/21 Nucleꢀ
odur 100ꢀ7 Cꢀ18 (Machery Nagel); solvent A: 5% acetonitrile
in water; solvent B: 95% acetonitrile in water; gradient: 75%
B to 100% B in 40 min, then 20 min with 100% B, flow rate
20 mL/min, to give 21.3 mg crude 1. A second preparative
HPLC using isocratic elution with 20% water in acetonitrile
for 40 min, then 20 min gradient to 95% acetonitrile, flow rate
20 mL/min, gave 15.6 mg pure product 1.
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Finally, we assessed the activity of hyaboron (1) in a numꢀ
ber of other biological assays (Table S3). In previous experiꢀ
ments, boron containing compounds were shown to display
various biological activities, including antibacterial, antifunꢀ
gal, antiviral, insecticidal, and cytotoxic effects.34 In addition
to its immuneꢀstimulatory activity in mammalian cells, hyaboꢀ
ron (1) displayed an antimicrobial effect (minimum inhibitory
concentration, MIC, in low µg/mL range) against Gramꢀ
positive bacteria and several yeasts and fungi. In addition, we
found 1 to inhibit the growth of various human cancer cell
lines with halfꢀinhibitory concentrations (IC50) in the nanomoꢀ
lar range. Most interestingly we found that 1 at a concentration
as low as 1 µM is a potent inhibitor of parasites that are the
causative agents of human African sleeping sickness, Chagas
disease, leishmaniasis and malaria, respectively.
Altogether, hyaboron and other boronated compounds likely
exhibit their activity via directly or indirectly acting as potasꢀ
sium ionophore.
Analytical HPLC: rt = 31.5 min; UV (methanol): λmax (log ε)
227 nm (4.62); [α]20 +56.2 (c = 1.0, CHCl3); IR (KBr) 3438,
D
2970, 2932, 1736, 1630, 1457, 1378, 1232, 1196, 1135, 1124,
1
1100, 1055, 1003, 948, 796, 701 cmꢀ1 ; H NMR data (700
MHz, CDCl3) δH 8.92 (br s, 2’’‒NHa), 8.26 (br s, 2’’‒NHb),
7.69 (d, J = 2.2 Hz, 13‒OH), 7.31 (m, 6’’‒H/8’’‒H), 7.27 (m,
5’’‒H/7’’‒H/9’’‒H), 6.67 (br s, 11’‒OH), 6.18 (m, 16’‒H),
6.16 (m, 15’‒H), 5.90 (dt, J = 14.2, 4.8 Hz, 14’‒H), 5.59 (m,
17‒H), 5.45 (dd, J = 11.4, 2.8 Hz, 16‒H), 5.32 (ddd, J = 11.0,
9.0, 6.0 Hz, 17’‒H), 5.19 (br d, J = 12.2 Hz, 2’’‒H), 5.10 (dt, J
= 6.0, 3.0 Hz, 11‒H), 5.04 (br d, J = 12.2 Hz, 9‒H), 5.02 (m,
20‒H), 4.89 (s, 2‒H), 4.83 (br s, 9’‒OH), 4.70 (m, 20’‒H),
4.68 (s, 2’‒H), 3.97 (m, 7‒H), 3.93 (m, 7’‒H), ), 3.79 (br t, J =
9.4 Hz, 11’‒H), 3.56 (t, J = 10.3 Hz, 9’‒H), 3.38 (s, 4‒OH),
3.28 (dd, J = 13.5, 12.2 Hz, 3’’‒Ha), 3.10 (m, 15‒H), 3.05 (br
d, J = 13.5 Hz, 3’’‒Hb), 2.94 (m, 18‒Ha), 2.88 (br s, 10’’‒H3),
2.81 (d, J = 2.8 Hz, 14‒H), 2.55 (m, 18’‒Ha), 2.39 (m, 13’‒
Ha), 2.16 (m, 13’‒Hb), 1.98 (m, 18’‒Hb), 1.95 (m, 18‒Hb), 1.86
(m, 19’‒Ha), 1.84 (br d, J = 14.4 Hz, 12‒Ha), 1.84 (m, 12’‒Ha),
1.82 (m, 4’‒H), 1.73 (m, 8’‒H), 1.68 (m, 5‒Ha, 10‒Ha, 19‒H2),
1.67 (ddd, J = 14.3, 11.5, 10.0 Hz, 10’‒Ha), 1.64 (m, 12’‒Hb),
1.57 (m, 12‒Hb, 5’‒H2), 1.45 (m, 5‒Hb), 1.38 (m, 6‒H2), 1.36
(m, 19’‒Hb), 1.35 (s, 22‒H3), 1.23 (br d, J = 14.3 Hz, 10’‒Hb),
1.23 (br d, J = 13.3 Hz, 10‒Hb), 1.22 (d, J = 6.2 Hz, 21‒H3),
1.16 (m, 8‒H), 1.09 (m, 6’‒H2), 1.07 (d, J = 6.2 Hz, 21’‒H3),
0.99 (d, J = 6.7 Hz, 22’‒H3), 0.77 (d, J = 7.5 Hz, 23’‒H3),0.76
(d, J = 7.5 Hz, 23‒H3); 13C NMR data (175 MHz, CDCl3) δC
173.5 (C, C‒1’), 173.4 (C, C‒1), 165.6 (C, C‒1’’), 135.6 (CH,
C‒14’), 135.1 (CH, C‒4’’), 134.5 (CH, C‒17), 131.2 (CH, C‒
16’), 129.8 (CH, C‒5’’/ C‒9’’), 128.7 (CH, C‒6’’/ C‒8’’),
128.1 (CH, C‒17’), 127.3 (CH, C‒7’’), 124.3 (CH, C‒16),
123.2 (CH, C‒15’), 104.8 (C, C‒3), 103.5 (C, C‒3’), 93.9 (C,
C‒13), 80.3 (CH, C‒9’), 78.0 (CH, C‒2), 77.2 (CH, C‒2’),
METHODS
General:
Optical rotation was measured on a PerkinꢀElmer 241 specꢀ
trometer, the IR spectrum on a Perkin Elmer FTꢀIR Spectrum
100 spectrometer and the UV spectrum on a Shimadzu UVꢀ
Vis spectrophotometer UVꢀ2450. NMR spectra were recorded
with Bruker Avance III 700, equipped with 5 mm TCI cryꢀ
oprobe (1H 700 MHz, 13C 175 MHz), and Bruker DMX 600
(1H 600 MHz, 13C 150 MHz) spectrometers. Chemical shifts δ
were referenced to the solvents chloroformꢀd (1H, δ = 7.27
ppm; 13C, δ = 77.0 ppm) and pyridineꢀd5 (1H, δ = 7.22 ppm;
13C, δ = 123.9 ppm. ESIMS spectra were acquired on an Amꢀ
azon ion trap mass spectrometer (Bruker Daltonik); HRESIMS
spectra were acquired on a Maxis timeꢀofꢀflight mass specꢀ
trometer (Bruker Daltonik), both combined with an Agilent
1200 series HPLCꢀUV system [column 2.1 × 50 mm, 1.7 ꢁm,
C18 Acquity UPLC BEH (Waters), solvent A: H2O + 0.1%
formic acid; solvent B: ACN + 0.1% formic acid, gradient: 5%
B for 0.5 min increasing to 100% B in 19.5 min, maintaining
100% B for 5 min, RF = 0.6 mL minꢀ1, UV detection 200ꢀ600
nm].
Fermentation:
Hyalangium minutum, strain Hym3, was cultivated on a 70
L scale in the presence of 1% Amberlite XADꢀ16 adsorber
resin (Rohm & Haas) in a BR150.3 reactor (100 L operating
volume, Chemap AG Switzerland / Bioreactor Baz Nr. 9ꢀ
4233) at 30 °C in a range of pH 7,1 ꢀ 7,3 (adjusted with 10 %
KOH / 5,0 % H2SO4) at 100 rpm, 0.05 vvm (5,0 NL/min),
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76.0 (CH, C‒11’), 72.1 (C, C‒4), 71.8 (CH, C‒7’), 71.5 (CH,
Minimum inhibitory concentrations (MIC) were determined
(Table S1) in 96ꢀwell microtiter plates in a serial dilution
assay with EBS medium as previously described.35
C‒11), 69.1 (CH, C‒20’), 68.4 (CH, C‒20), 68.3 (CH, C‒7),
61.59 (CH, C‒9), 61.56 (CH, C‒14), 59.9 (CH, C‒2’’), 52.1
(CH, C‒15), 43.2 (CH, C‒8’), 41.6 (CH, C‒8), 35.5 (CH2, C‒
19’), 34.63 (CH2, C‒5), 34.60 (CH2, C‒10’), 34.59 (CH2, C‒
19), 34.4 (CH, C‒4’), 33.9 (CH2, C‒12’), 33.2 (CH2, C‒12),
32.2 (CH3, C‒10’’), 32.0 (CH2, C‒10), 31.3 (CH2, C‒3’’), 30.5
(CH2, C‒6’), 29.0 (CH2, C‒5’), 28.7 (CH2, C‒13’), 25.2 (CH2,
C‒6), 25.0 (CH3, C‒22), 23.1 (CH2, C‒18’), 22.6 (CH2, C‒18),
20.7 (CH3, C‒21), 20.3 (CH3, C‒21’), 16.9 (CH3, C‒22’), 14.5
(CH3, C‒23’), 8.1 (CH3, C‒23); HRESIMS: m/z 1068.5710 [M
+ H]+ (calcd for C56H83BNO18 [M + H]+ 1068.5707).
Cytotoxicity assay
In vitro cytotoxicity (IC50) was determined against different
cell lines using our protocol described previously.35
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PBMCs isolation
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PBMCs were isolated from human buffy coats using standꢀ
ard protocol.33 In short, cells were separated by density gradiꢀ
ent centrifugation using Biocoll separating solution (Bioꢀ
chrom) followed by short erythrocyte lysis using BD pharm
Lyse.
Preparation of the (S)- and (R)-MTPA ester derivatives
of hyaboron (1)
For the (S)ꢀMTPA ester derivative hyaboron (1) (3.9 mg,
3.7 µmol) was dissolved in dry dichloromethane (350 µL) and
DMAP (3.9 mg), triethylamine (22 µL) and Rꢀ(ꢀ)ꢀMTPAꢀ
BMDMs preparation
o
Bone marrow was isolated from femur and tibia of either
wild type or Nlrp3 deficient C57BL/6 mice.14 Cells were filꢀ
tered and subjected to erythrocyte lysis. As described previꢀ
ously, bone marrow was differentiated in macrophages using
30% L929 supernatant in DMEM for seven days.36
chloride (22 µL) were added at 0 C and stirred under argon
atmosphere at room temperature for 24 h. The reaction mixꢀ
o
ture was quenched at 0 C with buffer solution (pH 7) and
extracted three times with dichloromethane, dried over MgSO4
and concentrated in vacuo to dryness. The crude product was
purified by silica gel chromatography (1 x 12.5 cm) using
tert.ꢀbutylꢀmethyl ether/nꢀheptane 2:3, then 1:1 as solvent. The
SꢀMTPA derivative 1a was further purified by preparative RPꢀ
HPLC chromatography on Nucleosil 100ꢀ7 (250 x 4) (Maꢀ
chery Nagel) using nꢀheptane/tert.ꢀbutylꢀmethyl ether 85:15
containing 1% methanol as solvent, flow rate 20 mL/min to
Primary Screening (iGLuc)
Primary screening was performed with J774 macrophages
stably expressing iGluc reporter system with constitutive
expression of NLRP3 under EF1α promotor.37 On the day of
the experiment, 30,000 cells were plated in each well in a 384ꢀ
well plate with the help of multidrop reagent dispenser (Therꢀ
mo Fisher Scientific). After 2 h, cells were stimulated with
natural compound library (NCL; DZIF) at 1 µM concentration.
Compounds were transferred onto the cells using high density
replication tool (with preꢀdetermined transfer capacity) in
Biomek FXP laboratory automation workstation (Beckman
Coulter). 2 h after stimulation, the gaussia signal was measꢀ
ured in the supernatant by adding coelenterazine at a final
concentration of 1µg/mL and measuring luminescence using
the Envision multilabel reader (Perkin Elmer).
1
yield 1.7 mg of compound 1a (m/z 1715.68). H NMR data
(600 MHz, CDCl3) of 1a: similar to 1, but δH 6.28 (15’‒H),
5.59 (14’‒H), 5.19 (11’‒H), 5.02 (9’‒H), 3.91 (7’‒H), 2.05
(13’‒Ha), 1.92 (10’‒H2), 1.75 (13’‒Hb), 1.75 (12’‒H2), 1.69
(8’‒H), 0.75 (23’‒H3).
The (R)ꢀMTPAꢀester of 1 was prepared analogously from
1
4.8 mg (4.5 µmol) of 1 to give 2.4 mg of compound 1b. H
NMR data (600 MHz, CDCl3) of 1b: similar to 1, but δH 6.32
(15’‒H), 5.70 (14’‒H), 5.22 (11’‒H), 5.09 (9’‒H), 3.72 (7’‒
H), 2.21 (13’‒Ha), 2.09 (10’‒Ha), 1.96 (12’‒H2), 1.95 (13’‒
Hb), 1.85 (10’‒Hb), 1.66 (8’‒H), 0.66 (23’‒H3).
Preparation and Analysis of L-FDAA-Derivatives
Stimulations
Hyaboron (1, 0.5 mg, 0.5 µmol) was dissolved in methanol
(150 µL) and hydrolyzed with 1 N sodium hydroxide (5 µL)
for 10 min. 1 N aqueous HCl (5 µL) was added and the reacꢀ
tion mixture diluted with 50 µL H2O. After addition of 1 N
NaHCO3 (5 µL) and 1% LꢀFDAA in acetone (20 µL) the reacꢀ
Human PBMCs were primed with 25 pg/ml of ultrapure
LPS from E. coli (Invivogen) for 4 h. Stimulation was perꢀ
formed with indicated concentrations of stimuli for 2 h. Mouse
BMDMs were primed with 200 ng/ml of LPS for 4 h followed
by stimulation with indicated stimuli for 8 h. For double
stranded DNA (dsDNA), pBlueScript plasmid DNA (1 µg/ml)
was complexed with Lipofectamine 2000 (Life Technologies)
according to the manufacturer’s protocol. In the high extracelꢀ
lular potassium experiment, medium was diluted with 150 mM
potassium chloride to the indicated potassium concentrations.
When indicated, the NLRP3 inhibitor MCC950 (10 µM) was
added in last hour of priming.
o
tion mixture was heated at 40 C for 1 h. The reaction was
quenched with 1 N HCl (5 µL) and evaporated to dryness. The
residue was dissolved in H2O (100 µL) and analyzed by RPꢀ
HPLCꢀMS using a gradient of 5% B to 50% B in 20 min (eluꢀ
ent A: H2O/CH3CN 95:5, 0.1% formic acid, eluent B:
CH3CN/H2O 95:5, 0.1% formic acid; flow: 0.6 mL/min); m/z
431.14; retention time (min) of FDAAꢀderivatized amino acid
standards (Bachem): NꢀMeꢀPheOH 14.0 min; NꢀMeꢀDꢀPheOH
14.1 min.
Immunoblotting and ELISA assay
For immunoblotting, 1x106 cells were plated per well in a
12ꢀwell plate and stimulations were performed as described
above. Supernatants were precipitated using methanolꢀ
Assays for Antimicrobial and Cytotoxic Activities
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chloroform extraction and immunoblotting was performed as
described previously.38 Primary antibodies for ILꢀ1β (AFꢀ401ꢀ
NA) and caspase 1 (AGꢀ20Bꢀ0042ꢀC100) were purchased
from R&D systems and Adipogen, respectively. Betaꢀactin
and secondary antibodies were purchased from Santa Cruz.
For human and mouse ILꢀ1β ELISA (BD Bioscience), 1x105
cell per well were plated in a 96ꢀwell flat bottom plate. After
stimulation, the cytokine ILꢀ1β was measured in the cellular
supernatant according to manufacturer’s instructions.
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ASSOCIATED CONTENT
Supporting Information. Complete experimental details and
spectral data is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* Email: hornung@genzentrum.lmu.de.
* Email: rom@helmholtzꢀhzi.de.
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1β Processing by Bioluminescence Sensors II: The iGLuc
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G., Hornung, V. (2011) Cutting edge: reactive oxygen species
inhibitors block priming, but not activation, of the NLRP3 inꢀ
flammasome. J. Immunol. 187, 613–617.
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ORCID
Frank Surup: 0000ꢀ0001ꢀ5234ꢀ8525
Eva Bartok: 0000ꢀ0003ꢀ0556ꢀ1950
Marc Stadler: 0000ꢀ0002ꢀ7284ꢀ8671
Rolf Müller: 0000ꢀ0002ꢀ1042ꢀ5665
Present addresses
∇
Bayer AG, Pharmaceuticals Research & Development, Frieꢀ
drichꢀEbertꢀStr. 217ꢀ333, 42096 Wuppertal, Germany.
○
Cargill GmbH, Seehafenstr. 2, 21079 Hamburg, Germany.
Author Contributions
+These authors made equal contributions.
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script.
Funding Sources
EB received financial support from BONFOR (University of
Bonn). DC and VH were supported by DZIF funds.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We kindly thank V. Dixit for Nlrp3ꢀdeficient mice (Genentech,
USA), C. Kakoschke for recording NMR spectra, A. Gollasch und
H. Steinmetz for MPLCꢀHRESIMS measurements, K. P. Conrad
for technical assistance, W. Collisi for conducting bioasꢀsays, K.
Mohr for the toc picture, W. Kessler and coꢀworkers for large
scale fermentations, M. Kaiser (Swiss TPH) for antiparasitic
assays, and Rolf Jansen for proofreading of the manuscript.
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ABBREVIATIONS
PRR, pathogen recognition receptors; PAMP, pathogen associated
molecular patterns; DAMP, danger associated molecular pattern;
NLR, NODꢀlike receptor; ASC, apoptosis associated speck like
protein containing caspase activation and recruitment domain; ILꢀ
1β, interleukinꢀ1β; PBMC, blood mononuclear cell.
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