Inhibition of chitin synthases and antifungal activities by 2′-benzoyloxycinnamaldehyde from Pleuropterus ciliinervis and its derivatives

Article abstract of DOI:10.1248/bpb.30.598

In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2′-benzoyloxycinnamaldehyde (2′-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2′-BCA inhibited chit

Full text of DOI:10.1248/bpb.30.598

5
98
Notes
Biol. Pharm. Bull. 30(3) 598—602 (2007)
Vol. 30, No. 3
Inhibition of Chitin Synthases and Antifungal Activities by
ꢀ-Benzoyloxycinnamaldehyde from Pleuropterus ciliinervis and Its
Derivatives
2
a
b
a
a
a
Tae Hoon KANG, Eui Il HWANG, Bong Sik YUN, Ki Duk PARK, Byoung Mog KWON,
c
,a
Chul Soo SHIN, and Sung Uk KIM*
a
b
Korea Research Institute of Bioscience and Biotechnology (KRIBB); Daejeon 305–806, Korea: Ginseng Science
c
Research Group, KT&G Central Research Institute; Daejeon 305–805, Korea: and Department of Biotechnology, College
of Engineering, Yonsei University; Seoul 120–749, Korea. Received September 5, 2006; accepted December 10, 2006
In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases
inhibitor, 2ꢀ-benzoyloxycinnamaldehyde (2ꢀ-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliin-
ervis NAKAI. 2ꢀ-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC s of 54.9 and
50
7
0.8 mg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 mg/ml. Its de-
rivatives, 2ꢀ-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory ac-
tivities than 2ꢀ-BCA, with the IC s of 37.2 and 26.6 mg/ml, respectively. Especially, the IC of compound VI
5
0
50
against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known
chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various
fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neo-
formans (MICꢁ16 mg/ml). Although the chemical synthesis of this compound has been reported, the present
study is the first report to describe the isolation of 2ꢀ-BCA from natural resources and chitin synthases in-
hibitory activities of its derivatives. These results suggested that 2ꢀ-BCA and its derivatives can potentially serve
as useful lead compounds for development of antifungal agents.
Key words Pleuropterus ciliinervis; 2ꢀ-benzoyloxycinnamaldehyde; chitin synthase inhibitor; antifungal activity
Fungal cells, like their bacterial and plant counterparts but synthase 1 is a nonessential repair enzyme of damaged
10)
unlike animal cells, are encased in the cell wall, which is chitin. Chitin synthase 3, on the other hand, is responsible
1)
essential for their survival. The fungal cell wall acts as an for the synthesis of chitin during bud emergence and growth,
12)
exoskeleton that provides the cell with resistance to tugor mating, and spore formation. As in S. cerevisiae, Candida
pressure, while maintaining their shape. As the most external albicans harbours three chitin synthases 1 (CaCHS1p), 2
structure of fungal cells, it is in direct contact with the exter- (CaCHS2p) and 3 (CaCHS3p), which are analogous to the S.
13)
nal medium, including the physiological defence mecha- cerevisiae chitin synthases 2, 1 and 3, respectively. There-
2)
nisms of the host. To ensure the continuous integrity of the fore, specific inhibitors of chitin synthase 2 and 3 from S.
cell wall without interfering with its plasticity and constant cerevisiae might be interesting leads to develop effective an-
growth, elaborate control mechanisms must be operating, tifungal agents.
which need to be strictly coordinated with those governing
The root of Pleuropterus ciliinervis NAKAI (Polygonaceae)
1)
the cell cycle. Accordingly, the biosynthesis of fungal cell has been used in a traditional Chinese folk medicine,
walls constitutes a good model for morphogenesis at the mo- “Hasuo”, which is used to treat inflammation, bacterial infec-
lecular level, and serve as a potential target in antifungal tions, suppurative dermatititis and gonorrhea in China as
3)
14,15)
chemotherapy because it is unique to the pathogen.
well as in Korea.
Stilbenes, anthraquinones, and flavoids
16)
Chitin, the b-(1,4)-linked homopolymer of N-acetyl-D-glu- have been isolated from the genus Pleuropterus. Recently,
cosamine (GlcNAc), is one of the most important structural a new naphthopyrone, pleuropyrone A with antioxidant activ-
17)
component of the cell walls of nearly all fungi and plays a ity was reported from Pleuropterus ciliinervis.
4)
major role in the determination of cell morphology. Al-
In the course of our continuing search for potent inhibitors
though the content of chitin in the cell wall varied among of chitin synthase 2 from the higher plants, a strong in-
5)
species, it is important for cell integrity. Chitin is absent hibitory compound against chitin synthase 2 of S. cerevisiae
from plant and mammalian species, while it is abundant in was found in the methanol extract of Pleuropterus ciliinervis,
6)
arthropods, most fungi, and other eukaryotes. Mutations which was identified as 2ꢀ-benzoyloxycinnamaldehyde (2ꢀ-
7)
that affect chitin synthesis cause osmotic sensitivity, abnor- BCA). Although this compound, which is synthesized by
mal morphology, aggregation, and growth arrest with elon- chemical synthesis, has been reported to demonstrate antitu-
8,9)
18,19)
gated buds. Thus, its biosynthesis has become a more and mor activity,
here, we describe the isolation of 2ꢀ-BCA
more attractive target for the design of antifungal agents. from the aerial parts of Pleuropterus ciliinervis and the syn-
Chitin is synthesized by chitin synthase 1 (ScCHS1p), 2 thesis of its derivatives, and their inhibitory activities against
(
ScCHS2p) and 3 (ScCHS3p) in Saccharomyces cere- chitin synthases of S. cerevisiae and various fungi including
9,10)
visiae,
for which the most comprehensive work including several human pathogenic fungi.
biochemical and genetic studies on fungal cell walls has been
carried out. Chitin synthase 2 is an essential enzyme for pri-
11)
mary septum formation and cell division, whereas chitin
∗
To whom correspondence should be addressed. e-mail: kimsu@kribb.re.kr
© 2007 Pharmaceutical Society of Japan

March 2007
599
MATERIALS AND METHODS
S. cerevisiae YPH499 and recombinant ECY38-38A strains
were cultivated at 30 °C overnight to reach the absorbance of
Strains and Culture Conditions The strains used in this 0.7 at 600 nm. Cells suspended in 50 mM Tris–HCl (pH 7.5)
amber
study were S. cerevisiae YPH499 (ura3-52 lys2-801
ade20101
(
containing 5 mM magnesium acetate were broken by vortex
trp1- D63 his- D200 leu2- D1), ECY38-38A mixing with glass beads. Cell walls were sedimented at
pAS6) (MATa chs1-23 chs2::LEU2 cal1/cds2 ura3-52 trp1- 4000ꢁg for 5 min and the supernatant fluid was centrifuged
ochre
20)
1
leu2-2 pAS6) and ECY38-38A (pWJC6) (MATa chs1-23 at 130000ꢁg for 45 min. The membrane pellet was sus-
21)
chs2::LEU2 cal1/cds2 ura3-52 trp1-1 leu2-2 pWJC6),
pended in the 50 mM Tris–HCl (pH 7.5) containing 33%
which were used as sources of ScCHS1p, 2p and 3p activity, glycerol used in the breakage, to a final volume of 1.6 ml/g
respectively. S. cerevisiae YPH499, the wild type for all three (wet weight) of cells.
synthases, was grown in YEPD [1% yeast extract, 2% Bacto
Chitin Synthases Assays The assays of chitin synthase
peptone (Difco), 2% glucose]. S. cerevisiae ECY38-38A 2 and 3 prepared from recombinant S. cerevisiae ECY38-
(
pAS6) and ECY38-38A (pWJC6), which can only overex- 38A (pAS6) and ECY38-38A (pWJC6), respectively, were
24)
press ScCHS2p and 3p, respectively, were grown in YPG conducted according to the method of Choi and Cabib.
[
1% yeast extract, 2% Bacto peptone, 2% galactose] at 30 °C. Chitin synthase 2 activity was measured by the procedure de-
24)
The strain was grown in Sabouraud dextrose medium at scribed previously. For the proteolytic activation step, reac-
0 °C. tion mixtures contained 32 mM Tris–HCl (pH 8.0), 1.6 mM
Chemicals Cinnamaldehyde (compound II) was pur- cobalt acetate, 1.0 mM UDP-[ C]-N-acetyl-D-glucosamine
3
1
4
chased from Sigma-Aldrich Chemical Co. (St. Louis, U.S.A.). (400000 cpm/mmol, NEN), 2 ml of trypsin at the optimal
1
4
Uridine diphosphate (UDP)-[U- C]-GlcNAc (400000 cpm/ concentration for activation (2.0 mg/ml), 20 ml of membrane
mmol) was purchased from NEN Life Science Products suspensions, and 14 ml of sample in a total volume of 46 ml.
(Boston, U.S.A.). All other reagents were of the highest The mixtures were incubated for 15 min at 30 °C. Proteolysis
grade available and used without further purification.
was stopped by adding 2 ml of a soybean trypsin inhibitor so-
Isolation of 2ꢀ-Benzoyloxycinnamaldehyde (I) The lution (4.0 mg/ml) at a concentration 2 times of the trypsin
aerial part of P. ciliinervis NAKAI was collected at Mt. De- solution used, and tubes were placed on ice for 10 min. N-
okyu, Muju, Korea. A voucher specimen has been deposited Acetyl-D-glucosamine was added to a final concentration of
under No. CFM-580 in the Korea Research Institute of Bio- 32 mM, and incubation at 30 °C was carried out for 90 min.
21,24)
science and Biotechnology. The aerial parts of P. ciliinervis For chitin synthase 3 activity,
2 kg) was extracted twice with 20 l of methanol at room for chitin synthase 2, except that 32 mM Tris–HCl (pH 7.5)
temperature for 5 d, filtered, and concentrated in vacuo to and 4.3 mM magnesium acetate were used. For the assay of
the assay was performed as
(
24)
yield a dark brown sticky solid (83 g). After solvent extrac- chitin synthase 1 activity, reaction mixtures contained 37
tion with ethyl acetate, the ethyl acetate layer (53 g) was sub- mM Tris–HCl (pH 7.5), 0.12% digitonin, 4.8 mM magnesium
jected to silica gel column chromatography (E. Merck, acetate, 2 ml of trypsin (1.0 mg/ml), 6 ml of membrane sus-
Kieselgel 60, 230—400 mesh, 7.5ꢁ35 cm) and eluted step- pension, and 14 ml of the test sample in a total volume of 41
wise with a gradient of n-hexane/ethyl acetate (5 : 5 to ethyl ml. After 15 min of incubation at 30 °C, 2 ml of trypsin in-
acetate only, v/v, each 6 l). The active fractions (5 : 5, v/v, hibitor (2.0 mg/ml) was added, and the tubes were placed on
4
00 mg) were subjected to Sephadex LH-20 column chro- ice. For the chitin synthase 2 and 3 assays, 32 mM N-acetyl-D-
1
4
matography (Amersham Bioscience, Sweden) with methanol glucosamine and 1.0 mM UDP-[ C]-N-acetyl-D-glucosamine
1.5ꢁ120 cm, 0.4 ml/min, each 8 ml). Crystallization was in- were added, and the mixtures were incubated for 30 min at
(
duced at active fractions (fraction 14—23). Crude crystal 30 °C. In all cases, the reaction was stopped by addition of
was resolved and finally purified by re-crystallization in 10% trichloroacetic acid and radioactivity of the insoluble
methanol, after which pure compound (100 mg) was col- chitin formed was counted after filtration through glass fiber
lected. Through the HPLC using an ODS column (Waters, filter (GF/C, Whatman). The concentration of protein was
2
5)
Xterra C , 5 mm, 250ꢁ4.6 mm), a single peak with the re- measured by the method of Lowry. Blank values were
1
8
tention time of 10.2 min was detected by a UV spectrometric measured with addition of 25% aqueous MeOH instead of
detector (254 nm). The column was then eluted with CH CN/ both enzyme and sample. Percent inhibition of chitin syn-
3
water (50 : 50 v/v) at a flow rate of 1.0 ml/min.
thase activity was calculated by substracting the blank values
Syntheses of 2ꢀ-BCA Derivatives 2ꢀ-BCA derivatives from both control and test sample values.
compound III—VI) were synthesized using the standard lit-
(

sample (cpm)ꢂ blank (cpm) 
erature methods. For the synthesis of 2ꢀ-substituted cin-
namaldehydes (III—VI), the commercially available 2ꢀ-sub-
stituted cinnamic acids were converted to 2ꢀ-substituted
methyl cinnamates by methylation with diazomethane, which
%
inhibitionꢃ 1ꢂ
ꢁ100
control (cpm)ꢂ blank (cpm)


The chitin synthase 1, 2, and 3 activity of the enzyme were
was prepared from Diazal. The methyl esters were reduced confirmed by positive control with polyoxin D and
by DIBAL-H (diisobutyl aluminum hydride) in tetrahydrofu- nikkomycin Z (Calbiochem. Co.) to compare the potency of
ran (THF) at ꢂ40 °C to give 2ꢀ-substituted cinnamyl alco- our compound against chitin synthases. Each isolated and
hols. Finally, the treatment of cinnamyl alcohols with MnO
gave 2ꢀ-cinnamaldehydes (III—VI).
control compounds were solubilized in 25% MeOH and dis-
tilled water to make a stock solution (1 mg/ml), respectively,
2
22)
Preparation of Crude Membranes Membranes of and an aliquot (14 ml) of the stock was used for each reaction
S. cerevisiae YPH499, ECY38-38A (pAS6) and ECY38-38A to give the final concentration of 280 mg/ml. The inhibitory
23)
(pWJC6) were prepared as described previously.
The activities were represented as average values in duplicates

6
00
Vol. 30, No. 3
C-NMR
1
13
obtained from two independent experiments. The preparation Table 1. H- and C-NMR Spectral Data of 2ꢀ-BCA
of microsome and chitin synthase 1 activity of C. albicans
1
13
2
6)
H-NMR
were conducted by the method described previously. For
CaCHS1p activity, the assay was performed the same as that
for ScCHS2p except that 32 mM Tris–HCl (pH 7.5) and 2 mM
cobalt chloride were used. Nikkomycin Z was used as a posi-
tive control for CaCHS1p inhibition. Each isolated and con-
trol compounds were solubilized in 25% dimethyl sulfoxide
Carbon No.
chemical shifts
chemical shifts
(ppm)
(ppm)
1
2
3
1
2
9.55 (1H, d, J=8 Hz)
6.78 (1H, q)
195.82
133.38
147.40
128.56
151.17
124.66
129.38
127.82
131.46
166.25
130.10
131.23
130.05
135.32
130.05
131.23
7.71 (1H, m)
ꢀ
ꢀ
3ꢀ
4ꢀ
ꢀ
6ꢀ
ꢄ
ꢄ
3ꢄ
4ꢄ
(
(
DMSO) and distilled water to make a stock solution
1 mg/ml), respectively.
7.30 (1H, dd, J =1 Hz)
7.55 (1H, m)
7.38 (1H, m)
7.85 (1H, dd, J=2 Hz)
Determination of Minimum Inhibitory Concentrations
MICs) MICs were determined by a two-fold serial broth
5
(
27)
microdilution method using Sabouraud dextrose broth. 2ꢀ-
BCA and its derivatives were dissolved in 25% MeOH, while
polyoxin D and nikkomycin Z were dissolved in distilled
water. The inoculum size of yeasts was 10 colony forming
unit (CFU)/ml. Antifungal activity was observed after 24 to
1
2
8.23 (1H, m)
7.58 (1H, m)
7.72 (1H, m)
7.58 (1H, m)
8.23 (1H, m)
3
5
6
7
ꢄ
ꢄ
ꢄ
4
8 h incubation at 30 °C. The MIC was defined as the lowest
concentration of antibiotics which completely inhibited the
growth of the organism when compared to a control well
containing no antibiotics.
RESULTS AND DISCUSSION
The bioactive compound from the aerial part of P. ciliin-
ervis was purified by solvent partition, silica gel and
Sephadex LH-20 column chromatographies, crystallization,
and HPLC. The structure of isolated compound was deter-
mined by EI-MS and various NMR spectroscopic analyses
1
13
1
1
including H, C, DEPT, H– H COSY, HMQC and HMBC.
1
The H-NMR (500 MHz, CD OD) of compound I (Table
3
1
9
6
) revealed an aldehyde proton appearing as doublets at d
.55 (Jꢃ8 Hz, H-1) and several aromatic protons (9H, d
.78—8.23). The multiple signals at d 8.23 (2H), 7.72 (1H)
and 7.58 (2H) were attributed to benzoyl hydrogens. Signals
at d 151.17, 131.46, 129.38, 128.56, 127.82 and 124.66 in
1
1
Fig. 1. H– H COSY and HMBC Connectivity for 2ꢀ-Benzoyloxycin-
1
3
the C-NMR (125 MHz, CD OD) data and those at d 133.38
3
namaldehyde and Its Derivatives
and 147.40, together with correlation indicated by the
HMBC spectrum between H-2 and H-3 with the carbonyl
group at 195.82, were typical of a 2ꢀ-hydroxylcinnamalde- and 2 of S. cerevisiae represented less inhibitory activities
hyde (Fig. 1). The signals at d 166.25, 135.32, 131.23 and than polyoxin D, whereas the inhibitory activity of the com-
1
7
30.05 were assigned to the benzoyl carbons from C-1ꢄ to C- pound against chitin synthase 2 showed 2.5-fold stronger in-
ꢄ, which are connected to 2ꢀ-hydroxyl. The presence of a hibitory activity than nikkomycin Z. In addition, this com-
benzoyl moiety in compound I was confirmed from peaks at pound showed more potent antifungal activities against sev-
m/z 105 and 77 in the EIMS. Structure analyses with EI-MS eral fungi such as S. cerevisiae, C. neoformans, C. lusitaniae,
ꢅ
spectrum (m/z 252, [M] ) and various NMR techniques in- and C. krusei (MICsꢃ16—64 mg/ml) than polyoxin D and
1
1
cluding H– H COSY, HMQC, and HMBC revealed that the nikkomycin Z, well-known chitin synthase inhibitors. Al-
isolated compound had molecular formular C H O and though polyoxin D and nikkomycin Z exhibited potent in-
1
6
12
3
was identified as 3-(2ꢀ-benzoyloxy phenyl)-propenal (2ꢀ-ben- hibitory activities against chitin synthases in assays per-
zoyloxycinnamaldehyde). The melting point of the com- formed in vitro, their antifungal activity would depend on the
pound was 78—80 °C.
permeability, because the compounds should penetrate into
Although several chitin synthase inhibitors have been iso- the fungal cell wall to exhibit antifungal activities through
2
8—32)
lated from microbes and higher plants,
yet been reported as chitin synthase inhibitor. The inhibitory cell membrane.
activities of this compound on chitin synthase isozymes were To determine whether structural changes of the benzoyl
2ꢀ-BCA has not the inhibition of chitin synthases, which are located on the
3
3,34)
1
4
investigated by the filter binding assay using UDP- C-Glc- group could enhance inhibitory activities, we synthesized
NAC as substrate. As shown in Table 2, 2ꢀ-BCA inhibited the cinnamaldehyde derivatives such as 2ꢀ-hydroxy- (III) and 2ꢀ-
chitin synthase 1 and 2 of S. cerevisiae in a dose-dependent methoxy- (IV) cinnamaldehyde, based on the previously re-
manner with IC values of 54.9 and 70.8 mg/ml, respectively, ported method. In addition, according to the report that the
but it showed no inhibitory activity on chitin synthase 3 up to incorporation of halogen atoms into a lead compound results
80 mg/ml. The IC50 values of 2ꢀ-BCA for chitin synthase 1 in analogues that are more lipophilic, we have synthesized
35)
5
0
2

March 2007
601
the derivatives with various halogen atoms such as 2ꢀ-chloro-
Considering that chemically synthesized 2ꢀ-BCA and its
(
V) and 2ꢀ-bromo- (VI) cinnamaldehyde. The inhibitory ac- derivatives have biological activity such as cytotoxicity
1
8,19,33)
tivities of compounds II (cinnamaldehyde), III and IV against human tumor cells,
other than differential in-
against chitin synthases showed weaker activities than 2ꢀ- hibitory activities on chitin synthase 1 and 2 of S. cerevisiae,
BCA (I), whereas compounds V and VI exhibited 1.9 and this compound and its derivatives may have multiple target
2.7-fold stronger inhibitory activities than 2ꢀ-BCA, respec- sites. Although the mode of action of 2ꢀ-BCA still remains to
tively (Table 2). Especially, the IC50 of compound VI against be investigated in the future, this is the first report to describe
chitin synthase 2 represented 1.7-fold more potent inhibitory the isolation of 2ꢀ-BCA from natural resources and its chitin
activity than polyoxin D. In addition, compound V and VI synthases inhibitory activities. This study also suggests that
exhibited 2 to 4 fold stronger antifungal activities, depending 2ꢀ-BCA and its derivatives may serve as useful lead com-
on the species of fungi, than 2ꢀ-BCA (Table 3). These results pounds for development of antifungal agents.
suggest that the significant structural changes leading to in-
crease inhibitory activities may be due to the introduction of
Acknowledgments We thank Dr. E. Cabib (NIH,
halogen atoms at 2ꢀ-hydroxyl position in place of a benzoyl Bethesda, MD, U.S.A.) and Dr. Hee-Moon Park (Chungnam
group. Interestingly, compounds V and VI showed the same National University, Daejeon, Korea) for providing the re-
antifungal activities as 2ꢀ-BCA against S. cerevisiae in spite combinant S. cerevisiae ECY38-38A and S. cerevisiae
of differential inhibitory activities on chitin synthase 2. Be- YPH499, respectively. This work was supported by a grant
sides, 2ꢀ-BCA, compound V and VI exhibited potent antifun- from the Plant Diversity Research Center of 21st Century
gal activities against human pathogenic fungi including Frontier Research program funded by the Ministry of Sci-
Cryptococcus and Candida species. The reason why these ence and Technology of the Korean government.
halogen compounds showed the increased antifungal activi-
ties might be their improved ability to penetrate the lipid
membrane in fungal cell. Based on these results, the antifun-
gal activities of 2ꢀ-BCA and its derivatives might be caused
at least in part by potent inhibitory activities for chitin syn-
thase 2 in S. cerevisiae. Besides we could not rule out the
possibility that the antifungal activities of these compounds
are due to the difference in permeability for the fungal cell
wall of various strains or due to the inhibition on other cellu-
lar targets. However, the detailed mode of action of the com-
pounds remains to be investigated.
REFERENCES
1)
Cabib E., Roh D. H., Schmidt M., Crotti L. B., Varma A., J. Biol.
Chem., 276, 19679—19682 (2001).
2) Sanz M., Carrano L., Jimenez C., Candian G., Trilla J. A., Duran A.,
Roncero C., Microbiology, 151, 2623—2636 (2005).
3
)
Smits G. J., Trilla J. A., Duran A., Roncero C., Microbiology, 147,
81—784 (2001).
Gooday G. W., J. Gen. Microbiol., 99, 1—11 (1977).
7
4)
5) Cabib E., Roberts S., Bowers B., Annu. Rev. Biochem., 51, 763—793
(1982).
6)
Ruiz-Herrera J., San-Blas G., Curr. Drug Targets Infect. Disord., 3,
7—91 (2003).
7
Table 2. Effect of 2ꢀ-BCA and Its Derivatives on Chitin Synthase
Isozymes
7
8
)
)
Bulawa C. E., Mol. Cell. Biol., 12, 1764—1776 (1992).
Bulawa C. E., Osmond B. C., Proc. Natl. Acad. Sci. U.S.A., 87, 7424—
7
428 (1990).
b)
Compounds (IC )
5
0
9) Shaw J. A., Mol P. C., Bowers B., Silverman S. J., Valdivieso M. H.,
Duran A., Cabib E., J. Cell. Biol., 114, 111—123 (1991).
0) Cabib E., Sburlati A., Bowers B., Silverman S. J., J. Cell. Biol., 108,
a)
Isozymes
c)
c)
I
II
III
IV
V
VI
PD
NZ
1
1
1
1
1
665—1672 (1989).
ScCHS1p
ScCHS2p
54.9 223.1 124.3 160.5 78.6 181.9
70.8 ꢆ280 84.1 89.9 37.2 26.6 46.2 175.6
3.1 1.0
3.7
0.6
1) Silverman S. J., Sbulati A., Slater M. L., Cabib E., Proc. Natl. Acad.
Sci. U.S.A., 85, 4735—4739 (1988).
2) Choi W. J., Sburlati A., Cabib E., Proc. Natl. Acad. Sci. U.S.A., 91,
ScCHS3p ꢆ280 ꢆ280 ꢆ280 ꢆ280 ꢆ280 >280
4
727—4730 (1994).
a) ScCHS1p was prepared from wild type S. cerevisiae YPH499. ScCHS2p: S. cere-
visiae ECY38-38A (pAS6) is a high-copy-number plasmid carrying CHS2 on a vector
containing a TRP marker. ScCHS3p: S. cerevisiae ECY38-38A (pWJC6) is a high-
copy-number plasmid carrying CAL/CSD2 (complete gene) under the control of the
GAL1 promoter. b) Unit: mg/ml. c) PD: polyoxin D, NZ: nikkomycin Z.
3) Mio T., Yabe T., Sudoh M., Satoh Y., Nakajima T., Arisawa M., Ya-
mada-Okabe H., J. Bacteriol., 178, 2416—2419 (1996).
14) Namba T., “The Encyclopedia of Wakan-Yaku (Traditional Sino-
Japanese Medicines) with Color Pictures,” Vol. 1, Hoikusha Publishing
Table 3. In Vitro Antifungal Activities of 2ꢀ-BCA and Its Derivatives against Various Fungi
b)
Compounds (MICs)
Fungi
c)
c)
I
II
III
IV
V
VI
PD
NZ
S. cerevisiae YPH499
16
32
32
32
64
64
64
128
128
ꢆ128
ꢆ128
ꢆ128
128
ꢆ128
ꢆ128
32
64
64
128
128
128
128
128
64
16
32
32
32
32
32
32
64
16
16
32
32
32
32
32
64
64
16
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
ꢆ100
S. cerevisiae ECY38-38A (pAS6)
S. cerevisiae ECY38-38A (pWJC6)
C. albicans ATCC10231
ꢆ128
ꢆ128
64
64
ꢆ128
32
128
ꢆ128
128
128
ꢆ128
128
a)
C. albicans A207
C. krusei ATCC6258
C. lusitaniae ATCC42720
C. tropicalis ATCC13803
C. neoformans ATCC36556
ꢆ100
ꢆ100
The experiment was repeated three times and MICs were shown as average values of three independent determinations. a) C. albicans A207: clinical isolate. b) Unit: mg/ml.
c) PD: polyoxin D, NZ: nikkomycin Z.

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