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Appl Environ Microbiol, February 1998, p. 714-720, Vol. 64, No. 2
Core Technology Area, Research, Novartis
Pharma Inc., CH-4002 Basel, Switzerland,1 and
Novartis Research Institute, A-1235 Vienna,
Austria2
Received 16 July 1997/Accepted 12 November 1997
Leptomycin B (LMB), a secondary metabolite produced by
Streptomyces sp. strain ATS 1287, with known antifungal and
antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits
significant antiproliferative activity. Since LMB itself turned out to
be distinctly cytotoxic, a bioconversion screening with a selected set
of 29 bacterial and 72 fungal strains was performed in order to obtain
metabolites of LMB with reduced antiproliferative effects. Several
derivatives of LMB, more polar than the parent compound and produced in
yields of >5%, were detected. Liquid chromatography-mass spectroscopy
analysis indicated the type of bioconversion. Fermentations (1-liter
scale) of those strains with high rates of transformation were suitable
for isolation and characterization of the most prominent metabolites.
Thus, bioconversion of LMB with Aspergillus flavus ATCC
9170 and Emericella unguis ATCC 13431 served for isolation
of the novel derivatives 26-hydroxy-LMB (30% was the concentration of
the metabolite [with respect to LMB] used for bioconversion) and
LMB-24-glutaminamide (90%), respectively. Streptomyces
rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%)
and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of
the LMB metabolites could be reduced through microbial conversion, none
of these metabolites inhibited the nuclear export of Rev better than
LMB itself.
Leptomycin B (LMB), produced by
Streptomyces sp. strain ATS 1287, has attracted attention
due to its antifungal (2-4) and antitumor (6, 7, 10,
18) effects. Recently it was found that, in addition to having
antiproliferative activity, LMB inhibits the nucleo-cytoplasmic
translocation of the human immunodeficiency virus type 1 (HIV-1)
regulatory protein Rev at low-nanomolar concentrations (16).
Rev protein, which is responsible for the cytoplasmic accumulation of
unspliced and singly spliced HIV-1 mRNA and thus for viral replication
(1), must be translocated from the nucleus to the cytoplasm
to exert its function (9, 15). Inhibitors of Rev protein
translocation may, therefore, be useful for HIV therapy
(16). However, due to its strong antiproliferative activity, LMB itself cannot be used therapeutically but, alternatively, may serve
as a tool for dissecting nuclear transport pathways (16).
The aim of this study was to find LMB derivatives with reduced
antiproliferative activities but preserved levels of inhibition of the
nuclear export of Rev. Bioconversion reactions, such as hydroxylation
and conjugation, are relevant detoxification reactions in living beings
(14). Therefore, microbial conversion can serve as a tool to
effect these useful derivation reactions with cellular enzyme
equipment.
To identify novel metabolites of LMB, screening with a large variety of
microorganisms selected on the basis of previous bioconversions was
performed. Biotransformation products were detected by analyzing a
combination of high-performance liquid chromatography (HPLC)-UV and
liquid chromatography-mass spectroscopy (LC-MS) measurements. Fermentations (11) of those strains with high rates of
transformation served for the isolation of derivatives, and their
structures were elucidated mainly by nuclear magnetic resonance (NMR)
techniques. All characterized metabolites of LMB were tested for
their antiproliferative effects and inhibition of Rev
translocation.
(A preliminary account of some of this work was presented at a poster
session at the Biocat Screening '96 Symposium, Ede, The Netherlands,
15 to 18 December 1996.)
General experimental section.
For HPLC analysis, we used a
model D-6000A interface; a model AS-2000A autosampler; a model L-6200A
pump; a model L-4500 diode array detector; a model T-6300 column
thermostat; software from Merck and Hitachi; a Lichrocart 125-4, Superspher 100, RP-18 column (4 µm); an RP-18 precolumn (5 µm); a
linear gradient of 1% H3PO4-CH3CN (30 to 100% CH3CN) for 9 min, and a flow rate of 1.5 ml/min. Biotransformation products were detected at 220 nm by HPLC
combined with diode array detection. For HPLC isolation, we used linear
gradients of 0.098% H3PO4 (pH
4)-CH3CN (30 to 55% CH3CN) for 45 min and
0.098% H3PO4 (pH 4)-CH3CN (55 to
80% CH3CN) for 15 min, a Spherisorb RP-18 column (5 µm)
(250 by 20 mm), UV detection at 220 nm, and a flow rate of 18 ml/min.
To determine retention times for LC-MS, electrospray ionization
MS was performed on a Finnigan TSQ 7000 triple-quadrupole MS. The
instrument was controlled and data were analyzed with ICIS software
(Finnigan). The electrostatic-spray ion source was operated at 4.5 kV,
and the atmosphere-vacuum transfer capillary was heated at 220°C. The
column effluent was split 5:1 with a Valco tee, allowing a flow rate of
0.05 ml/min into the electrospray nebulizer. Full-scan mass spectra
were recorded from mass-to-charge ratios (m/z) of 500 to
1,400 in 1.8 s for the MS analysis of the derivatives. For LC
analysis, we used a Spherisorb 5, RP-18 column (5 µm) and a gradient
system with a column temperature of 50°C, UV detection at 220 nm, and
a flow rate of 0.25 ml/min. All NMR spectra were recorded at 20°C on
a Bruker AMX-400 spectrometer equipped with a 5-mm-diameter inverse
triple-resonance probe. The spectra were acquired with
CDCl3 by using trimethylsilyl (TMS) as the internal
reference. Resonance assignments were obtained from a series of
homonuclear and heteronuclear two-dimensional experiments.
Chemical shifts are given in the Microorganisms and bioconversion conditions.
Strains
(72 fungi, 29 bacteria) were purchased from the American Type Culture
Collection, the National Collection of Industrial and Marine Bacteria,
the Northern Utilization Research and Development Division, and the
Centraalbureau voor Schimmelcultures.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Microbial Conversion Products of
Leptomycin B
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
scale; J values are
given in hertz. MS spectra were recorded on an MS, model VC 70-SE
(positive fast atom bombardment [FAB+]; Xe, 8 keV), with nitrobenzylic alcohol plus LiI as the matrix and an acceleration voltage of 8 kV.
25°C. Spores and cells of one
agar culture were suspended in 10 ml of 0.9% NaCl.
Two-hundred-microliter Erlenmeyer flasks each containing 50 ml of seed
culture medium (see below) were inoculated with 2 ml of these
suspensions and incubated on a rotary shaker (200 rpm) at 24°C
(fungi) or 27°C (bacteria) for 4 days. One-hundred-microliter
Erlenmeyer flasks each containing 25 ml of main culture medium (see
below) were inoculated with 2.5 ml of the seed culture and incubated on
a rotary shaker (200 rpm) at 24°C. After 24 h, the parent
compound dissolved in methanol (MeOH) was added to a final
concentration of 50 mg/liter to the main cultures, which were harvested
after 48, 72, or 96 h of incubation. All controls were treated
under the same fermentation conditions as described for the
bioconversion samples. Compound controls containing 50 mg of LMB/liter
of main culture medium and strain controls lacking LMB were prepared. To stabilize LMB, main culture media that were adjusted to pHs of 2, 3, 4, 5, 6, 7, 8, 9, and 10 and contained 50 mg of LMB/liter of medium
were prepared and treated as described above for the compound controls.
Compositions of the liquid seed media. For bacteria, we used medium 231, which consists of 0.005% CaCO3, 0.7% glucose, 0.45% yeast extract (Gistex), 0.5% malt extract liquid (Wander), 1.0% soluble starch, 0.25% N-Z-Amine type A (Sheffield), and 0.1% (vol/vol) trace element solution a (pH 7.0). Trace element solution a contains 0.01% H3BO3, 0.5% FeSO4 · 7H2O, 0.005% KI, 0.2% CoCl2 · 6H2O, 0.02% CuSO4 · 5H2O, 0.2% MnCl2 · 4H2O, 0.4% ZnSO4 · 7H2O, and 0.1% (vol/vol) H2SO4 (97%). For fungi, we used medium SA, which consists of 0.1% Bacto Agar, 0.4% yeast extract (Gistex), and 2.0% malt extract liquid (Wander) (pH 5.0 to 5.5), and medium SB, which consists of 0.75% soy protein (Siber & Hegner), 2.0% dextrose (Difco), 0.1% malt extract liquid (Wander), 0.1% brewer's yeast (Cenovis), 0.05% KH2PO4, 0.005% MgSO4 · 7H2O, 0.002% CaCl2 · 2H2O, 0.001% NaCl, 0.1% (vol/vol) trace element solution b, and 0.1% Bacto Agar (pH 6.0 to 6.2). Trace element solution b consists of 0.003% Na2MoO4 · 2H2O, 0.44% ZnSO4 · 7H2O, 0.55% FeSO4 · 7H2O, 0.008% CuSO4 · 5H2O, 0.018% MnCl2 · 4H2O, and 0.2% (vol/vol) H2SO4 (97%).
Compositions of the main media. For bacteria, we used medium Act1, which consists of 0.5% glucose, 1.5% soluble starch, 1.0% N-Z-Amine type A (Sheffield), 0.2% brewer's yeast (Cenovis), 0.06% K2HPO4-0.003% KH2PO4, 0.01% MgSO4 · 7H2O, 0.01% CaCl2 · 2H2O, 0.005% NaCl, and 0.1% (vol/vol) trace element solution a (pH 6.2 to 6.5), and medium Act3, which consists of 0.5% Bacto Tryptone, 0.3% Bacto yeast extract, and 1.0% glucose (pH 7.1). For fungi, we used medium MA, which consists of 2.0% glucose, 0.2% soy protein (Siber & Hegner) 0.2% malt extract liquid (Wander), 0.2% yeast extract (Gistex), 0.2% KH2PO4, 0.05% MgSO4 · 7H2O, 0.1% (vol/vol) trace element solution b, and 0.1% Bacto Agar (pH 5.1 to 5.4), medium MB, which consists of 1.0% soy protein (Siber & Hegner), 3.0% dextrose (Difco), 0.2% malt extract (Difco), 0.2% brewer's yeast (Cenovis), 0.075% KH2PO4, 0.001% MgSO4 · 7H2O, 0.005% CaCl2 · 2H2O, 0.002% NaCl, and 0.15% (vol/vol) trace element solution b (pH 6.0 to 6.2), and medium MC, which consists of 0.6%, (NH4)2HPO4, 0.3% KH2PO4, 0.001% NaCl, 0.01% MgSO4 · 7H2O, 0.002% CaCl2 · 6H2O, and 0.05% (vol/vol) trace element solution b (pH 7.0 to 7.2). For agar slants, we used medium 330, which consists of 1.8% Bacto Agar, (vol/vol) 0.02% trace element solution a, 0.2% yeast extract (Bacto), and 1.0% soluble starch (pH 7.0).
Extraction and HPLC analysis. Ethyl acetate (EtOAc; 20 ml) was added to 20 ml of the main culture, shaken for 20 min at 24°C at 200 rpm, and centrifuged at 4,500 rpm for 10 min. The EtOAc layer (15 ml) was evaporated to dryness and redissolved in 1.5 ml of MeOH. These extracts (10 µl) were then chromatographed by HPLC (for the conditions, see "General experimental section").
Isolation of the LMB biotransformation products. One-liter culture broths of the strains ATCC 9170, ATCC 28893, ATCC 13431, and ATCC 55060, to which 50 mg of LMB/liter was added, were filtered, and the filtrates were extracted three times with EtOAc (700, 500, and 400 ml), washed with demineralized water, and dried with Na2SO4. These extracts were each separated in one step by preparative HPLC (for the conditions, see "General experimental section"). Fractions (18 ml) were monitored for the desired leptomycin derivatives by analytical HPLC (for conditions, see "General experimental section"). The enriched fractions were extracted with equal volumes of EtOAc, and the extracts were dried under vaccuum. The extracts of the culture broths of ATCC 9170 (75 mg), ATCC 28893 (109.8 mg), and ATCC 13431 (81.3 mg) yielded compounds 2 (7 mg), 3 (6 mg), 4 (12 mg), and 5 (17 mg).
26-Hydroxy-LMB (compound 2).
UV (MeOH), c = 0.1 g/liter; 
max (in nanometers), 225. Infrared (IR) (film)
max (per centimeter), 3,352, 2,966, 2,930, 1,707, 1,644, 1,455, 1,374, 1,252, 1,100, 1,046, 968, 885, 825, 736, 703. FAB-MS (positive
mode), [M+Li-H]Li+, 569 (m/z 86);
[M+Li]+, 563 (m/z 100), 551 (m/z
16), 425 (m/z 42), 413 (m/z 32), 397 (m/z 53), 377 (m/z 35). For 1H-NMR
(CDCl3), see Table 2.
4,11-Dihydroxy-LMB (compound 3).
UV (MeOH), c = 0.1 g/liter; max (in nanometers), 227. IR (film) max
(per centimeter), 3,350, 2,963, 2,928, 1,708, 1,456, 1,376, 1,260, 1,099, 969, 870, 823, 736. FAB-MS (positive mode), [M+Li-H]Li+, 585 (m/z 63);
[M+Li]+, 579 (m/z 47), 473 (m/z
24), 466 (m/z 100), 460 (m/z 27), 447 (m/z 72), 398 (m/z 35), 355 (m/z 35),
328 (m/z 82). For 1H-NMR (CDCl3),
see Table 2.
2,3-Dihydro-LMB (compound 4).
IR (film) max (per
centimeter), 2,964, 2,930, 1,706, 1,641, 1,455, 1,375, 1,250, 1,136, 966, 867, 702, 636, 620. FAB-MS (positive mode),
[M+Li-H]Li+, 555 (m/z 83);
[M+Li]+, 549 (m/z 63), 417 (m/z
22), 405 (m/z 31), 399 (m/z 100), 393 (m/z 34), 333 (m/z 20). For 1H-NMR
and 1H-1H-COSY (CDCl3), see Table
2.
LMB-24-glutaminamide (compound 5).
IR (film) max (per
centimeter), 3,348, 2,966, 2,931, 1,708, 1,663, 1,528, 1,455, 1,374, 1,286, 1,248, 1,480, 1,101, 1,046, 970, 825. FAB-MS (positive mode),
[M+Li-H]Li+, 681, (m/z 100),
[M+Li]+, 675 (m/z 76), 629 (m/z
11), 593, (m/z 11), 572, (m/z 12), 419 (m/z 13), 397 (m/z 27). For 1H-NMR,
InvHCCORR, and 1H-1H COSY (CDCl3),
see Table 3.
Biological assays. Inhibition of Rev translocation and antiproliferative activity of LMB metabolites were determined by means of the Rev translocation assay (RTA) (16) and the sulforhodamine B staining assay for cellular protein (13), respectively.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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In the course of a screening of 29 bacterial and 72 fungal strains, various derivatives of LMB were detected (Table 1). Stability experiments with LMB under those fermentation conditions chosen for bioconversion cultures showed unambiguously that LMB remains stable from pH 4 to 8. As a consequence of this and with respect to strain and compound controls, which were treated exactly like the biotransformation samples, all products described in this paper are bioconversion metabolites.
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One-liter fermentations of strains exhibiting high rates of transformation served for the isolation of the conversion derivatives 2 to 5 by preparative HPLC (Fig. 1).
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Through the screening, Streptomyces aureofaciens ATCC 13304 (5), Saccharopolyspora erythraea ATCC 11635 (12), Aspergillus fischeri ATCC 1020 (5), and Aspergillus flavus ATCC 9170 (5) were found to convert LMB into metabolite 2 (Table 1). Among these strains Aspergillus flavus ATCC 9170, an already known bioconversion strain exhibiting hydrolase activity (5), which produced predominantly metabolite 2 (Fig. 2), served for its isolation. The similarity between the UV spectrum of metabolite 2 and that of the parent compound confirmed the detection of an LMB derivative (Fig. 3).
|
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An m/z of 556 for compound 2 indicated monohydroxylation of
LMB. Whereas most of the signals in the 1H-NMR analysis of
compound 2 appeared almost identical to those of compound 1, a
quadruplet of a single proton at 4.58 ppm was observed instead of
the multiplet of the two H-26s at 2.2 ppm in the spectrum of
compound 1 (Table 2). The loss of one
proton at C-26 and the simultaneous significant downfield shift of the remaining H-26 is a strong hint for the introduction of a hydroxyl group. Concomitantly, the signal of the neighboring 27-CH3
changed from a triplet at 1.06 ppm in the spectrum of compound 1 to a doublet at 1.36 ppm, supporting this assumption. By these facts,
compound 2 was identified as the novel 26-hydroxy-LMB (Fig. 1).
|
Streptomyces rimosus ATCC 28893 and Mucor
hiemalis ATCC 20095, strains already known for their oxidation
capabilities (8, 17), transformed compound 1 into the polar
metabolite 3. An m/z of 572 indicated a dihydroxylation of
compound 1. The 1H-NMR spectrum of compound 3 lacked the
signal of H-4, and in contrast to the spectrum of compound 1 (4-CH3 = 0.96 ppm, d), the signal of the
4-CH3 appeared as a singlet at 1.34 ppm. This result
indicated the presence of a hydroxyl group at C-4. The second
hydroxyl group is located at C-11, since a multiplet of a single proton
at 4.58 ppm was observed instead of the multiplet of the two
protons of C-11 at 2.03 ppm (Table 2). Based on the evaluation of
the 1H-NMR data, compound 3 was characterized as
4,11-dihydroxy-LMB (Fig. 1), a new metabolite. However, the relative
stereochemistries of the newly generated hydroxyl groups bearing chiral
centers in metabolites 2 and 3 were not determined.
Another metabolite of Streptomyces rimosus ATCC 28893 is the
novel 2,3-dihydro-LMB (compound 4), which differs in mass from compound
1 by 2 U. This metabolite was also detected through bioconversion with
Gliocladium catenulatum ATCC 10523 and Aspergillus
alliaceus ATCC 10060. In the 1H NMR spectrum of
compound 4, reduction of the olefin was clearly demonstrated by the
disappearance of the significant olefinic signals of H-2 at 6.00 ppm and H-3 at 6.95 ppm and the appearance of H-2 and H-3 proton
signals in a range typical for saturated hydrocarbons (H-2, [2H] 2.61 ppm, m; H-3a, [1H] 1.69 ppm, m; H-3b, [1H] 1.98 ppm,
m). The assignment of the H-2 and H-3 protons was achieved by
1H-1H COSY (Fig. 3), showing a coupling of the
H-3a and -3b with the H-2 (a and b) and the H-4. Based on these data,
compound 4 was identified as the novel 2,3-dihydro-LMB (Fig. 1).
Finally, Emericella unguis ATCC 13431 (5) and
Streptomyces aureofaciens ATCC 13189 (5) produced
metabolite 5, an enzymatic conjugation product. First hints were
obtained from the MS spectrum, which showed an increase in mass of 128 U with respect to that of compound 1. The proton signal pattern typical
for LMB remained unchanged in the 1H NMR spectrum of
compound 5, but additional signals were observed (Table
3). 1H-1H COSY
(Table 3) and HCCORR spectra (Table 3) revealed that compound 5 is an
amide formed by condensation of the free carboxyl group of LMB with the
amino group of glutamine. The new broad signal at 7.28 ppm could be
assigned to the amide proton, which showed a prominent coupling with
the new H-2'. However, the relative stereochemistry of the chiral C-2'
in the new glutaminamide was not determined. According to these data,
compound 5 was identified as LMB-24-glutaminamide.
|
Biological data. The microbial transformation metabolites of LMB were evaluated in an RTA (16) and a 72-h proliferation assay with HeLa-Rev cells (16) (Table 4). Different types of enzymatic modifications (hydroxylation, reduction, and conjugation) at different sites of the molecule, achieved by microbial conversion, led to decreases in activities in both assays with respect to that of LMB. The loss of RTA activity was most significant for metabolite 4, indicating that the enone system has to be preserved for the inhibition of Rev translocation. All compounds described showed a close correlation in their activities in both the RTA and the proliferation assay. So far no derivative, either chemically (11) or enzymatically modified, with a therapeutic window between Rev translocation and cytotoxicity has been found.
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ACKNOWLEDGMENTS |
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We thank L. Gschwind and M. Uhl for technical support and for preparing the large-scale fermentations and E. Bürgin for the LC-MS measurements. We are grateful to H. U. Naegeli for his advice in isolation of metabolites and to H. U. Gremlich and J. France for their help in the elucidation of the structures of the described bioconversion products. We finally thank E. Schreiner and M. Grassberger for critically reviewing the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Core Technology Area, Research, Novartis Pharma Inc., CH-4002 Basel, Switzerland. Phone: 41-61-324-3594. Fax: 41-61-324-3279. E-mail: JeanJacques.Sanglier{at}Pharma.Novartis.com.
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Abstract
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Materials & Methods
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References
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Appl Environ Microbiol, February 1998, p. 714-720, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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