Sinefungin VA and dehydrosinefungin V, new antitrypanosomal antibiotics produced by Streptomyces sp. K05-0178

Article abstract of DOI:10.1038/ja.2010.102

Two new nucleotide antibiotics, named sinefungin VA and dehydrosinefungin V, were separated by cation exchange column chromatography and purified by HPLC from the culture broth of Streptomyces sp. K05-0178, together with the known antibiotics, sinefungin, dehydrosinefungin and KSA-9342. The structures of the two novel sinefungin analogs were elucidated by spectroscopic studies, including various NMR and advanced peptide chemical methods. Sinefungin VA consists of adenosine and ornithylvalylalanine, whereas dehydrosinefungin V consists of 4′,5′-dehydroadenosine and ornithylvaline. Sinefungin VA showed potent antitrypanosomal activity with an IC₅₀ value of 0.0026 g ml 1 in vitro without cytotoxicity against MRC-5 cells. Dehydrosinefungin V showed moderate antitrypanosomal activity (IC₅₀=0.15 μg ml^-1).

Full text of DOI:10.1038/ja.2010.102

The Journal of Antibiotics (2010) 63, 673–679
2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10 $32.00
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ORIGINAL ARTICLE
Sinefungin VA and dehydrosinefungin V, new
antitrypanosomal antibiotics produced by
Streptomyces sp. K05-0178
Megumi Niitsuma1, Junko Hashida1, Masato Iwatsuki1, Mihoko Mori2, Aki Ishiyama1, Miyuki Namatame1,
Aki Nishihara-Tsukashima1, Atsuko Matsumoto2, Yoko Takahashi2, Haruki Yamada2, Kazuhiko Otoguro1,
2
2
¯
Kazuro Shiomi and Satoshi Omura
Two new nucleotide antibiotics, named sinefungin VA and dehydrosinefungin V, were separated by cation exchange column
chromatography and purified by HPLC from the culture broth of Streptomyces sp. K05-0178, together with the known
antibiotics, sinefungin, dehydrosinefungin and KSA-9342. The structures of the two novel sinefungin analogs were elucidated
by spectroscopic studies, including various NMR and advanced peptide chemical methods. Sinefungin VA consists of adenosine
and ornithylvalylalanine, whereas dehydrosinefungin V consists of 4¢,5¢-dehydroadenosine and ornithylvaline. Sinefungin VA
showed potent antitrypanosomal activity with an IC50 value of 0.0026 lg mlꢀ1 in vitro without cytotoxicity against MRC-5 cells.
Dehydrosinefungin V showed moderate antitrypanosomal activity (IC50¼0.15 lg mlꢀ1).
The Journal of Antibiotics (2010) 63, 673–679; doi:10.1038/ja.2010.102; published online 22 September 2010
Keywords: antitrypanosomal antibiotics; dehydrosinefungin V; sinefungin VA; Streptomyces
INTRODUCTION
were isolated from a culture broth of Streptomyces sp. K05-0178
Human African trypanosomiasis, also known as sleeping sickness, is (Figure 1). In this paper, the fermentation, isolation, physicochemical
regarded as one of the major neglected tropical diseases. It is a properties and structure elucidation and biological properties of 1 and
complex disease caused by infection with protozoan Trypanosoma 2 are described.
spp. parasites, transmitted through the bites of infected tsetse flies.
There are two forms of the disease, each having two distinct stages, RESULTS
and without treatment the disease is fatal. Today, some 70 million Taxonomy of the producing strain K05-0178
people are exposed to the threat of human African trypanosomiasis.1 The strain K05-0178 was isolated from a soil sample collected at
It was estimated that, in 2004, approximately 50000–70000 new cases Minato-ku, Tokyo, Japan. The vegetative mycelia developed well on
of human African trypanosomiasis were occurring annually.1 Cur- yeast extract–malt extract agar, oatmeal agar and inorganic salts–
rently, only four drugs, pentamidine, suramin, melarsoprol and starch agar and did not show fragmentation into coccoid forms or
eflornithine, are registered for the treatment of human African bacillary elements. The color of vegetative mycelia showed red. Aerial
trypanosomiasis. All four are unsatisfactory, as they cannot be mycelia were produced abundantly on inorganic salts–starch agar and
administered orally and are hampered by severe toxicity and increas- the aerial mass color showed pink. The strain formed verticillate
ing resistance of the parasites. Although there is a clear and urgent sporophores bearing terminal umbels of spore chains (Figure 2). The
need for new classes of antitrypanosomal compounds, and despite the mature spore chain had 5–10 spores per chain. The spores were
recent introduction of a new treatment using a combination of two cylindrical in shape, 1.0ꢁ0.5 mm in size and had a smooth surface.
existing drugs, no effective new drugs have been introduced into the Brownish soluble pigment was produced on yeast extract–malt extract
field for 20 years.
agar. The isomer of diaminopimelic acid in whole-cell hydrolysates of
During our screening program to discover new antitrypanosomal strain K05-0178 was determined to be the LL-form. An almost
antibiotics, two novel sinefungin analogs, sinefungin VA (1) and complete 16S rDNA sequence (1400 nucleotides) was determined,
dehydrosinefungin V (2), and three recognized structurally related and the result of the identification analysis by EzTaxon.org server
compounds, sinefungin (3), dehydrosinefungin (4) and KSA-9342 (5), version 2.1 (http://147.47.212.35:8080/) showed the highest similarity
1Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan and 2Kitasato Institute for Life Sciences and Graduate School of
Infection Control Sciences, Kitasato University, Tokyo, Japan
¯
Correspondence: Professor K Shiomi or Professor S Omura, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1
Shirokane, Minato-ku, Tokyo 108-8641, Japan.
E-mail: shiomi@lisci.kitasato-u.ac.jp or omuras@insti.kitasato-u.ac.jp
Received 26 April 2010; revised 23 July 2010; accepted 12 August 2010; published online 22 September 2010

New antitrypanosomal sinefungin VA and dehydrosinefungin V
M Niitsuma et al
674
Figure 1 Structures of sinefungin VA (1), dehydrosinefungin V (2), sinefungin (3), dehydrosinefungin (4) and KSA-9342 (5).
buffers (pH 3.8, 180 ml each). The 0.4 and 0.6 M CH3COONH4
fractions were neutralized by NH4OH, desalted by the Micro Acylizer
and concentrated in vacuo to dryness to yield a brown material
(1143 mg). The material was dissolved in a small amount of water
and purified by HPLC on an XBridge Shield RP18 column (10 i.d.
ꢁ250 mm, Waters Milford, MA, USA) with 1.5% CH3CN/10mM
CH3COONH4 (pH 10) at 4.0 ml minꢀ1 detected at UV 210 nm. The
retention times of sinefungin VA (1), dehydrosinefungin V (2) and
KSA-9342 (5) were 33, 42 and 55min, respectively (Figure 3a). Each
solution was neutralized by HCl, desalted by Sephadex G-10 (GE
Healthcare, Buckinghamshire, UK) gel filtration chromatography and
concentrated in vacuo to dryness to afford 1 (0.2mg), 2 (29 mg) and 5
(49 mg) as pale brown powders. On the other hand, the 0.2 M
CH3COONH4 fraction was neutralized, desalted by the Micro Acylizer
and concentrated in vacuo to dryness to yield a brown material
(438mg). It was dissolved in a small amount of water and purified
by HPLC on an XBridge Shield RP18 column (10 i.d. ꢁ250 mm) with
0.5% CH3CN/10 mM CH3COONH4 (pH 10) at 4.0 mlminꢀ1 detected
at UV 210 nm. The retention times of sinefungin (3) and dehydrosi-
Figure 2 Scanning electron micrograph of the strain K05-0178 grown on
1/10 V-8 juice agar at 27 1C for 18 days. Bar represents 10 mm.
value with Streptomyces coerulescens NBRC 12758T (AB184122, 99.6%) nefungin (4) were 9 and 13min, respectively (Figure 3b). Each
and Streptomyces abikoensis NBRC 13860T (AB184537, 99.6%). On the solution was neutralized by HCl, desalted by the Micro Acylizer and
basis of the morphological and cultural properties and 16S rDNA concentrated in vacuo to dryness to afford 3 (73mg) and 4 (60 mg) as
sequence similarity, the strain was identified with whorl-forming pale brown powders.
Streptomyces species and designated Streptomyces sp. K05-0178.2,3
Physicochemical properties
The physicochemical properties of sinefungin VA (1) and dehydrosi-
Isolation
The procedure for isolation of sinefungin analogs is summarized in nefungin V (2) are summarized in Table 1. They are both readily
Scheme 1. The 6-day-old culture broth (10 l) was extracted with 10l of soluble in water, methanol and acetone. They showed absorption
ethanol and filtered through a paper to separate into supernatant and maxima at 206 and 260 nm in UV spectra. The broad IR absorption at
mycelia. The supernatant was concentrated under reduced pressure to 3300cmꢀ1 suggested the presence of hydroxyl groups. The similarity
remove ethanol and then passed through a Dowex 50Wꢁ2 [H+] in physicochemical properties strongly suggested that they are struc-
column (50 i.d. ꢁ400 mm, Dow Chemical, Midland, MI, USA) turally related to sinefungin (3).
previously activated. After washing with water (600ml), the active
materials were eluted with 1.0 M NH4OH (600 ml), followed by Identification of sinefungins, dehydrosinefungin and KSA-9342
neutralization by HCl and desalted by a Micro Acylizer S3 The molecular formulae of 3–5 were elucidated by HR-FAB-MS to be
(ASTOM, Tokyo, Japan). The whole eluate was applied on a Toyopearl C15H23N7O5, C15H21N7O5 and C23H35N9O7, respectively (Table 1).
SP-650C column (3.0 i.d. ꢁ300 mm, Tosoh Co., Tokyo, Japan) The analysis of 1H, 13C and 2D NMR spectra data led to the
previously equilibrated with 0.1 M CH3COONH4 buffer (pH 3.8). identification of 3–5 as sinefungin,4 dehydrosinefungin5 and KSA-
After washing with 0.1 M CH3COONH4 buffer (pH 3.8, 180 ml), the 9342,6 respectively. As the assignments of their chemical shifts have
active materials were eluted with 0.2, 0.4 and 0.6 M CH3COONH4 not been reported, the data are shown in Table 2.
The Journal of Antibiotics

New antitrypanosomal sinefungin VA and dehydrosinefungin V
M Niitsuma et al
675
Scheme 1 Purification of sinefungin analogs.
Figure 3 Chromatographic profile of (a) sinefungin VA (1), dehydrosinefungin V (2) and KSA-9342 (5) and (b) sinefungin (3) and dehydrosinefungin (4) by
preparative HPLC.
Structure elucidation of sinefungin VA (1)
The molecular formula of 1 was elucidated by HR-FAB-MS to in HMBC revealed the C-1 of the sinefungin moiety was attached to
presence of the valylalanine moiety. The correlations from H-2¢ to C-1
1
be C23H37N9O7, requiring 10 degrees of unsaturation. The H and the amino group of valylalanyl moiety, to form an amide bond. This
13C NMR spectral data of 1 are listed in Table 2. The 13C NMR and was confirmed by fragment ions of m/z 463 [M–Ala+H]+ and m/z 346
heteronuclear multiple quantum coherence (HMQC) spectra indi- [M–Val-Ala+H]+. The absolute configurations of amino acid residues
cated 23 carbons, which were classified into one carboxyl carbon at dc of 1 were elucidated to be L-Val and L-Ala by HPLC analysis of
181.3, two amide carbonyl carbons, five olefinic carbons, one hemi- the hydrolysates of 1 using a chiral column. Thus, the structure of
aminal carbon at dc 88.8, three oxygenated sp3 methine carbons, four 1 is shown in Figure 1, and the compound was designated as
nitrogenated sp3 methine carbons, one sp3 methine carbon, three sp3 sinefungin VA.
methylene carbons and three methyl carbons. The characteristic UV
spectra and fragment ion (m/z 135) in FAB-MS indicated the presence Structure elucidation of dehydrosinefungin V (2)
of an adenine moiety. Moreover, the spin systems from H-2 to H-10, The molecular formula of 2 was elucidated by HR-FAB-MS to be
1
deduced by the COSY, and the correlations from H-2 to C-1 and C-3, C20H30N8O6, requiring 10 degrees of unsaturation. The H and 13C
from H-5 to C-7, from H-8 and H-9 to C-10, and from H-10 to C-11 NMR spectral data of 2 are listed in Table 2. The 13C NMR and
and C-15 of the adenine moiety in the heteronuclear multiple-bond HMQC spectra indicated 20 carbons, which were classified into one
correlation (HMBC) spectra, revealed 1 was a sinefungin analog carboxyl carbon at dc 178.4, one amide carbonyl carbon at dc 169.1,
(Figure 4). This was confirmed by comparison with the NMR spectra seven olefinic carbons containing one oxygenated olefinic carbon at dc
of sinefungin (Table 2). The spin systems from H-2¢ to H3-5¢ and from 159.9, one hemiaminal carbon at dc 87.9, two oxygenated sp3 methine
H-200 to H3-300 by the COSYand the correlations from H-2¢ and H-200 carbons, three nitrogenated sp3 methine carbons, one sp3 methine
to C-1¢ and from H3-300 to C-100 in the HMBC spectra indicated the carbon, two sp3 methylene carbons and two methyl carbons. The
The Journal of Antibiotics

New antitrypanosomal sinefungin VA and dehydrosinefungin V
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676
Table 1 Physicochemical properties of sinefungin analogs
Sinefungin VA (1)
Dehydrosinefungin V (2)
Sinefungin (3)
Dehydrosinefungin (4)
KSA-9342 (5)
Appearance
Pale brown powder
Pale brown powder
C20H30N8O6
478
Pale brown powder
C15H23N7O5
381
Pale brown powder
C15H21N7O5
379
Pale brown powder
C23H35N9O7
549
Molecular formula
Molecular weight
C23H37N9O7
551
FAB-MS (m/z)
Positive
552 [M+H]+
574 [M+Na]+
479 [M+H]+
523 [M-H+2Na]+
382 [M+H]+
380 [M+H]+
550 [M+H]+
572 [M+Na]+
HR-FAB-MS (m/z)
Calcd.
552.2894
523.2005
382.1839
380.1682
572.2557
Found
552.2889
523.2031
382.1843
380.1552
572.2555
50%MeOH
UV lmax
nm (e)
206, 261
206, 259
206, 260
206, 259
206, 259
cmꢀ1
3459, 1652, 1402
3428, 1640, 1407
3434, 1650, 1405
3434, 1621, 1403
3428, 1644, 1400
KBr
IR nmax
Solubility
Soluble
H2O, MeOH, acetone
H2O, MeOH, acetone
H2O, MeOH, acetone
H2O, MeOH, acetone
H2O, MeOH, acetone
EtOAc, CHCl3
Insoluble
n-Hexane, CHCl3
n-Hexane, CHCl3
n-Hexane, CHCl3
n-Hexane, CHCl3
Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; HR-FAB-MS, high-resolution FAB-MS; IR, infrared; UV, ultraviolet.
Table 2 1H and 13C nuclear magnetic resonance spectral date of sinefungin analogs
Sinefungin VA (1)
Dehydrosinefungin V (2)
Sinefungin (3)
Dehydrosinefungin (4)
KSA-9342 (5)
Position 13C
1H (J in Hz)
13C
1H (J in Hz)
13C
1H (J in Hz)
13C
1H (J in Hz)
13C
1H (J in Hz)
1
2
3
169.7
52.7
27.3
—
169.1
52.5
27.6
—
173.8
53.8
22.8
—
174.0
53.9
26.8
—
171.0
52.7
28.0
—
4.17 (1H, m)
1.38 (1H, m)
1.82 (1H, m)
1.40 (1H, m)
1.84 (1H, m)
3.58 (1H, m)
2.20 (2H, m)
4.03 (1H, m)
1.17 (1H, m)
1.77 (1H, m)
1.61 (1H, m)
1.79 (1H, m)
4.10 (1H, m)
4.92 (1H, d, 3.1)
—
3.62 (1H, t, 7.0)
1.80 (2H, m)
3.80 (1H, t, 7.0)
1.88 (1H, m)
1.92 (1H, m)
1.90 (1H, m)
1.95 (1H, m)
4.20 (1H, m)
5.05 (1H, m)
—
4.02 (1H, m)
1.22 (1H, m)
1.84 (1H, m)
1.65 (1H, m)
1.86 (1H, m)
4.12 (1H, m)
4.80 (1H, m)
—
4
28.2
28.2
26.5
1.70 (1H, m)
1.90 (1H, m)
3.41 (1H, m)
2.06 (2H, m)
28.7
29.0
5
6
49.0
34.2
48.6
99.5
48.8
34.0
48.5
99.5
48.5
99.5
7
73.3 4.35 (1H, dt, 7.2, 7.1) 159.9
80.0 4.27 (1H, m) 67.4
73.2 4.80 (1H, dd, 7.3, 6.2) 72.5
72.5 4.15 (1H, dt, 6.2, 6.0) 159.6
79.5 4.20 (1H, dd, 6.2, 6.2) 67.2
160.5
67.3
8
4.94 (1H, d, 3.1)
4.89 (1H, m)
6.20 (1H, d, 7.3)
5.10 (1H, d, 6.2)
4.67 (1H, m)
9
72.3 4.60 (1H, dd, 7.0, 6.2) 72.3 5.00 (1H, dd, 7.0, 6.2) 72.4 4.99 (1H, dd, 7.5, 6.2)
10
88.8
6.05 (1H, d, 7.3)
87.9
88.5
5.88 (1H, d, 7.0)
87.8
6.23 (1H, d, 7.0)
88.0
6.17 (1H, d, 7.5)
Adenine
11
141.0
119.5
155.9
153.2
149.2
8.25 (1H, s)
140.7
119.5
155.9
153.1
149.0
8.16 (1H, s)
140.5
119.2
155.8
153.0
148.5
8.10 (1H, s)
140.8
119.4
155.6
152.7
150.6
8.30 (1H, s)
141.0
119.3
156.0
153.5
149.2
8.26 (1H, s)
12
—
—
—
—
—
13
—
8.24 (1H, s)
—
—
8.05 (1H, s)
—
—
8.08 (1H, s)
—
—
8.24 (1H, s)
—
—
8.11 (1H, s)
—
14
15
Valine
1¢
172.2
60.2
30.3
18.6
17.7
—
178.4
61.8
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
172.0
60.5
30.0
17.7
18.1
—
2¢
4.16 (1H, d, 7.0)
2.15 (1H, m)
3.91 (1H, d, 7.0)
4.05 (1H, d, 7.0)
2.07 (1H, m)
3¢
30.2 1.95 (1H, dqq, 7.2, 7.0)
4¢
0.96 (3H, d, 7.2)
0.98 (3H, d, 7.2)
18.9
17.8
0.81 (3H, d, 7.2)
0.78 (3H, d, 7.2)
0.88 (3H, d, 7.0)
0.87 (3H, d, 7.0)
5¢
Alanine
100
200
181.3
51.3
17.9
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
179.2
50.5
17.5
—
4.09 (1H, q, 7.1)
1.31 (3H, d, 7.1)
4.01 (1H, q, 7.2)
1.22 (3H, d, 7.2)
300
The Journal of Antibiotics

New antitrypanosomal sinefungin VA and dehydrosinefungin V
M Niitsuma et al
677
Figure 4 COSY and HMBC studies of sinefungin VA (1) and dehydrosinefungin V (2).
Table 3 Antitrypanosomal activity and cytotoxicity of sinefungin
analogs
However, 1 exhibited significantly less cytotoxicity against MRC-5
cells than sinefungin. Dehydrosinefungin V (2) showed moderate
antitrypanosomal activity with IC50 values of 0.14mg mlꢀ1, which
was similar to that of dehydrosinefungin (4) and KSA-9342 (5).
A selectivity index (see Table 3), created to allow direct comparisons
of effectiveness and cytotoxicity, indicated that sinefungin VA was
almost 200-fold less potent than sinefungin but that, significantly, it
was some 30-fold better than suramin.
IC50(mg mlꢀ1
)
Antitrypanosomal activity Cytotoxicity Selectivity index
Compound
T. b. b. GUTat 3.1
MRC-5
MRC-5/T. b. b.
Sinefungin VA (1)
Dehydrosinefungin V (2)
Sinefungin (3)
0.0026
0.147
45
7.96
79
41923
54
DISCUSSION
0.00021
0.0587
0.186
376 000
35
Sinefungin and its analog, dehydrosinefungin, were isolated from a
culture broth of Streptomyces griseolus and found to have antifungal
and antitrypanosomal properties.4,5 Sinefungin is a nucleoside anti-
biotic containing an ornithine residue linked by a C–C bond to the 5¢
carbon of adenosine. The biosynthetic precursors of sinefungin were
reported as being L-arginine and ATP, using incorporation studies as
well as HPLC analysis,7 although the details remain unclear. The
isolation of sinefungin and its various analogs from the same culture
broth in this study might provide a clue to help solve their biosynth-
Dehydrosinefungin (4)
KSA-9342 (5)
Suramina
2.10
3.91
4100
21
1.58
463
Abbreviations: IC50, inhibitory constant 50; T.b.b., Trypanosoma brucei brucei.
aSuramin is a drug used to treat Human African Trypanosomiasis.
characteristic
UV spectrum and fragment ion (m/z 135) in FAB mass spectrum esis pathway. Sinefungin is a structural analog of S-adenosylmethio-
indicated the presence of an adenine moiety. Moreover, the spin nine, a molecule that has an important role in the biosynthesis
systems from H-2 to H-6 and from H-8 to H-10, deduced by the of polyamines and in transmethylations of proteins and lipids.
1H-1H COSY, and the correlations from H-2 to C-1 and C-3, from Sinefungin and dehydrosinefungin are very potent inhibitors of
H-6 to C-7 and C-8, from H-8 to C-10 and from H-10 to C-11 and S-adenosylmethionine-dependent transmethylations.8 Sinefungin was
C-15 of the adenine moiety in the HMBC spectra, revealed that 2 was also reported to exhibit strong antiviral, antileishmanial and anti-
a dehydrosinefungin analog (Figure 4). This was confirmed by tumor activity.8–10 In our study, compounds having a single bond
comparison with the NMR spectra of dehydrosinefungin (Table 2). between C-6 and C-7 of adenosine (sinefungin and sinefungin VA)
The spin systems from H-2¢ to H3-5¢ by the COSYand the correlations showed potent antitrypanosomal activity as compared with com-
from H-2¢ to C-1¢ in the HMBC spectra indicated the presence of a pounds having a double bond (dehydrosinefungin, dehydrosinefungin
valyl moiety. The correlations from H-2¢ to C-1 in HMBC revealed the Vand KSA-9342). Many biological activities of sinefungin are believed
C-1 of dehydrosinefungin was attached to the amino group of the valyl to be related to the inhibition of S-adenosylmethionine-dependent
moiety to form an amide bond. The absolute configuration of the methyltransferase enzymes.11–16
valine residue was determined to be the L-form by HPLC analysis of
When administered intraperitoneally, sinefungin cures mice
the hydrolysates of 2 using a chiral column. The structure of 2 is infected with T. brucei brucei, Trypanosoma congolense and Trypano-
shown in Figure 1, and the compound was designated as dehydrosi- soma vivax.17,18 It was well tolerated in mice, and when the dose per
nefungin V.
injection was 0.05–5mg kgꢀ1, sinefungin was highly effective against
African trypanosomes. Sinefungin and dehydrosinefungin were also
leishmanicidal at concentrations ranging from 0.13 to 2.6mM in vitro.9
Biological activities
As shown in Table 3, sinefungin VA (1) showed potent activity against Our data confirm that sinefungin is both a potent antitrypanosomal
Trypanosoma brucei brucei GUTat 3.1 strain with an IC50 value of and that it has a moderate cytotoxic effect against MRC-5 cells.
2.60ꢁ10ꢀ3 mg mlꢀ1, which was 10-fold less than that of sinefungin Hitherto, sinefungin has not been developed because of its serious
(IC50 value of 2.10ꢁ10ꢀ4 mg mlꢀ1) but approximately 600-fold side effects (nephrotoxicity at subcurative levels in goats and dogs and
higher than that of the commonly used therapeutic drug, suramin. toxicity for bone marrow cells).19,20 However, synthetic studies for
The Journal of Antibiotics

New antitrypanosomal sinefungin VA and dehydrosinefungin V
M Niitsuma et al
678
some analogs have been reported to improve the therapeutic (2.0–2.5ꢁ104 trypanosomes mlꢀ1) was transferred in a 96-well microtiter plate and
index.21,22 Our results indicate that sinefungin VA retains potent
antitrypanosomal properties but with a greatly reduced cytotoxicity,
significantly outperforming suramin, indicating that it could be
a promising candidate for development into a novel new anti-
trypanosomal drug.
5.0 ml of a test compound solution (dissolved in 5.0% DMSO) was added and
incubated for 72 h at 37 1C (long incubation–low inoculation test). After that, 10 ml
of the fluorescent dye alamer blue was added to each well. After incubation for
3–6 h, the resulting solution was read at 528/20 nm excitation wavelengths and 590/
30 nm emission wavelengths by an FLx800 fluorescence microplate reader (BioTek
Instruments, Winooski, VT, USA). The IC50 values were determined using the
fluorescent plate reader software (KC-4, BioTek). Successive subcultures were done
in 24-well tissue culture plates under the same conditions.
METHODS
Taxonomic studies
We observed cultural and physiological characteristics of strain K05-0178
grown on yeast extract–malt extract agar, oatmeal agar and inorganic salts–
starch agar, after incubation for 2 weeks at 271C. The morphological properties
were observed with a scanning electron microscope (model JSM-5600, JEOL,
Akishima, Japan) after cultivation on 1/10 V8 juice agar at 271C for 18 days.
Isomers of diaminopimelic acid in whole-cell hydrolysates were determined by
TLC, following the standard methods of Becker et al.23 and Hasegawa et al.24
16S rDNA was amplified by PCR and sequenced directly on an ABI model 377A
automatic DNA sequencer using a PRISM Ready Reaction Dye Primer Cycle
Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA).
Cytotoxic activity in vitro
Measurement of cytotoxic activity against human fetal lung fibroblast MRC-5
cells was carried out as described previously.26
ACKNOWLEDGEMENTS
This study was supported, in part, by funds from the Drugs for Neglected
Diseases initiative, Quality Assurance Framework of Higher Education from the
Ministry of Education, Culture, Sports, Science and Technology in Japan, and
All Kitasato Project Study. We are grateful to Ms Hitomi Sekiguchi and Mr
Toshiaki Furusawa for their technical assistance, Dr Hiroshi Honma and Dr
Masae Sekine, School of Pharmacy, Kitasato University for amino acid analysis,
and Ms Akiko Nakagawa, Dr Kenichiro Nagai and Ms Noriko Sato, School of
Pharmacy, Kitasato University for measurements of mass and NMR spectra.
Fermentation
The strain K05-0178 was grown and maintained on agar slants consisting of
1.0% starch, 0.3% NZ amine, 0.1% yeast extract, 0.1% meat extract, 1.2% agar
and 0.3% CaCO3. A loop of spores of strain K05-0178 was inoculated into
100ml of seed medium, consisting of 2.4% starch, 0.1% glucose, 0.3% peptone,
0.3% meat extract, 0.5% yeast extract and 0.4% CaCO3 (adjusted to pH 7.0
before sterilization) in a 500-ml Erlenmeyer flask. The flask was incubated on a
rotary shaker (210 r.p.m.) at 271C for 3 days. A 1 ml portion of the seed culture
was transferred to 500-ml Erlenmeyer flasks (total 100) containing 100ml of
production medium, consisting of 0.5% glucose, 0.5% corn steep powder,
1.0% oat meal, 1.0% Pharmamedia (Traders Protein, Lubbock, TX, USA),
1
2
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259 (2006).
Williams, S. T., Goodfellow, M. & Alderson, G. Genus Streptomyces Waksman and
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3
Witt, D. & Stackebrandt, E. Unification of the genera Streptoverticillium and Strepto-
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0.5% K2HPO4, 0.5% MgSO4ꢂ7H2O, 5.0ꢁ10ꢀ4% FeSO4ꢂ7H2O, 5.0ꢁ10ꢀ4
%
MgCl2ꢂ4H2O, 5.0ꢁ10ꢀ4
%
CuSO4ꢂ5H2O and 5.0ꢁ10ꢀ4
%
(adjusted to pH 7.0 before sterilization) and fermentation was carried out on
CoCl2ꢂ6H2O
4
5
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I. Discovery and isolation. J. Antibiot. 26, 463–465 (1973).
Berry, D. R. & Abbott, B. J. Incorporation of carbon-14-labeled compounds into
sinefungin (A9145), a nucleoside antifungal antibiotic. J. Antibiot. 31, 185–191
(1978).
a rotary shaker (210r.p.m.) at 27 1C for 6 days.
6
Yamashita, K., Suzuki, A., Isogai, A., Sawai, N. & Kobayashi, R. Fungicidal antibiotic
KSA-9342 and its manufacture with Streptoverticillium. Japan Patent Application
63-297398, 5 December (1988).
Malina, H., Tempete, C. & Robert-Gero, M. Biosynthesis of sinefungin by cell-free
extract of Streptomyces incarnates NRRL 8089. J. Antibiot. 40, 505–511 (1987).
Vedel, M., Lawrence, F., Robert-Gero, M. & Lederer, E. The antifungal antibiotic
sinefungin as a very active inhibitor of methyltransferases and of the transformation
of chick embryo fibroblasts by Rous sarcoma virus. Biochem. Biophys. Res. Commun.
85, 371–376 (1978).
General experiments
NMR spectra were measured using a Varian XL-400 spectrometer or a
Varian Inova 600 spectrometer (Varian, Palo Alto, CA, USA), with 1H NMR
at 400 or 600 MHz and 13C NMR at 100 or 150 MHz in D2O. The chemical
shifts are expressed in p.p.m. and are referenced to HDO (4.76 p.p.m.) in the
1H NMR spectra and the end of both fields (0, 200 p.p.m.) in the 13C NMR
spectra. FAB-MS spectra were measured on a JEOL JMS AX-505 HA mass
spectrometer. IR spectra (KBr) were taken on a Horiba FT-210 Fourier (Horiba,
Kyoto, Japan) transform infrared spectrometer. UV spectra were measured with
a Beckman DU640 spectrophotometer (Beckman, Fullerton, CA, USA). Optical
rotation was measured on a JASCO model DIP-181 polarimeter (JASCO,
Hachioji, Japan).
7
8
9
Paolantonacci, P., Lawrence, F., Lederer, F. & Robert-Gero, M. Differential effect
of sinefungin and its analogs on the multiplication of three Leishmania species.
Antimicrob. Agents Chemother. 28, 528–531 (1985).
10 Suhadolnik, R. J. Nucleosides as Biological Probes 19–23 (Wiley, New York, 1979).
11 Pugh, C. S. & Borchardt, R. T. Effects of S-adenosylhomocysteine analogues on
vaccinia viral messenger ribonucleic acid synthesis and methylation. Biochemistry
21, 1535–1541 (1982).
12 Fuller, R. W. & Nagarajan, R. Inhibition of methyltransferases by some new analogs of
S-adenosylhomocysteine. Biochem. Pharmacol. 27, 1981–1983 (1978).
13 MaCammon, M. T. & Parks, L. W. Inhibition of sterol transmethylation by S-adeno-
sylhomocysteine analogs. J. Bacteriol. 145, 106–112 (1981).
14 Bacchi, C. J. et al. Fate of soluble methionine in African trypanosomes: effects of
metabolic inhibitors. Biochem. J. 309, 737–743 (1995).
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mRNA(guanine-7-)-methyltransferase, mRNA(nucleoside-2¢-)-methyltransferase, and
viral multiplication. J. Biol. Chem. 253, 4075–4077 (1978).
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(1996).
17 Dube, D. K., Mpimbaza, G., Allison, A. C., Lederer, E. & Rovis, L. Antitrypanosomal
activity of sinefungin. Am. J. Trop. Med. Hyg. 32, 31–33 (1983).
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the treatment of Trypanosoma (Nannomonas) congolense infections in goats. Trop.
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Amino acid analysis
Each of 1–3 (100 mg) was completely hydrolyzed in a gas phase of 6 M HCl
(100ml) at 110 1C for 18h in a reaction vial in which air was replaced by N2 gas,
using the Pico-Tag work station (Waters). For elucidation of the absolute
configuration of amino acids, the hydrolysates were dissolved in distilled water
and subjected to HPLC analysis (Column, Sumichiral OA-5000 (4.6 i.d.
ꢁ150mm, Sumika Chemical Analysis Service, Osaka, Japan); Mobile Phase,
2 mM CuSO4; flow rate, 1.0mlminꢀ1; detection, 254nm; column temperature,
40 1C). Retention time (min): L-Ala 5.02, D-Ala 7.36, L-Val 12.2 and D-Val 22.1.
Antitrypanosomal activity in vitro
In vitro antitrypanosomal activities against T. brucei brucei strain GUTat 3.1
were measured, using the method described previously.25 In brief, the strain
GUTat 3.1 was cultured in Iscove’s modified Dulbecco’s medium (IMDM) with
various supplements and 10% heat-inactivated fetal bovine serum at 371C,
under 5.0% CO2/95% air. Subsequently, 95 ml of the trypanosomes suspension
20 Barton, D. H. et al. Total synthesis of uracil analogues of sinefungin. J. Med. Chem. 35,
63–67 (1992).
The Journal of Antibiotics

New antitrypanosomal sinefungin VA and dehydrosinefungin V
M Niitsuma et al
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21 Lyga, J. W. & Secrist, J. A. III. Synthesis of chain-extended and C-6¢ functionalized
precursors of the nucleoside antibiotic sinefungin. J. Org. Chem. 48, 1982–1988 (1983).
22 Blanchard, P. et al. Synthesis and biological activity of sinefungin analogues. J. Med.
Chem. 34, 2798–2803 (1991).
23 Becker, B., Lechevalier, M. P. & Lechevalier, H. A. Chemical composition of cell-wall
preparation from strains of various form-genera of aerobic actinomycetes. Appl.
Microbiol. 13, 236–243 (1965).
24 Hasegawa, T., Takizawa, M. & Tanida, S. A rapid analysis for chemical
grouping of aerobic actinomycetes. J. Gen. Appl. Microbiol. 29, 319–322
(1983).
25 Otoguro, K. et al. Selective and potent in vitro antitrypanosomal activities of 10
microbial metabolites. J. Antibiot. 61, 372–378 (2008).
26 Otoguro, K. et al. Potent antimalarial activities of polyether antibiotic, X-206.
J. Antibiot. 54, 658–663 (2001).
The Journal of Antibiotics