Piperazine propanol derivative as a novel antifungal targeting 1,3-β-D-glucan synthase

Article abstract of DOI:10.1248/bpb.28.2138

1,3-β-D-Glucan synthase, which synthesizes a main component of fungal cell wall, is one of the promising targets for antifungal agents. In order to identify novel chemical classes of 1,3-β-D-glucan synthase inhibitors, we screened a chemical library monit

Full text of DOI:10.1248/bpb.28.2138

2138
Notes
Biol. Pharm. Bull. 28(11) 2138—2141 (2005)
Vol. 28, No. 11
Piperazine Propanol Derivative as a Novel Antifungal Targeting
b
1,3- -D-Glucan Synthase
Osamu KONDOH,*,a,b Yukiko INAGAKI,a,b Hiroshi FUKUDA,a,b Eisaku MIZUGUCHI,a,b Yoshikazu OHYA,c
b
Mikio ARISAWA,a,b Nobuo SHIMMA,a,b Yuko AOKI,a,b Masahiro SAKAITANI,a,b and Takahide WATANABE
a Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd. (formerly Nippon Roche Research Center); 200
Kajiwara, Kamakura, Kanagawa 247–8530, Japan: b Nippon Roche Research Center; 200 Kajiwara, Kamakura,
Kanagawa 247–8530, Japan: and c Department of Integrated Biosciences, Graduate School of Frontier Science, University
of Tokyo; Bldg. FSB-101, 5–1–5 Kashiwanoha, Kashiwa, Chiba 277–8562, Japan.
Received May 5, 2005; accepted August 25, 2005; published online September 7, 2005
1,3-b-D-Glucan synthase, which synthesizes a main component of fungal cell wall, is one of the promising
targets for antifungal agents. In order to identify novel chemical classes of 1,3-b-D-glucan synthase inhibitors, we
screened a chemical library monitoring inhibition of the Candida albicans 1,3-b-D-glucan synthase activity. The
piperazine propanol derivative GSI578 [(2,6-difluoro-phenyl)-carbamic acid 3-(4-benzothiazol-2-yl-piperazine-1-
yl)-propyl ester] was identified as a potent inhibitor against 1,3-b-D-glucan synthase with an IC50 value of
0.16 mM. GSI578 exhibited in vitro antifungal activity against pathogenic fungi including C. albicans and As-
pergillus fumigatus. Temperature-sensitive mutations of the FKS1 gene in the Dfks2 background of Saccha-
romyces cerevisiae, where FKS1 and FKS2 encode putative catalytic subunits of 1,3-b-D-glucan synthase, altered
sensitivity to GSI578. This suggests that the antifungal activity of the piperazine propanol derivative has an effect
on 1,3-b-D-glucan synthase inhibition. Results of our initial evaluation suggest that the piperazine propanol de-
rivative is a novel chemical structure of the class of antifungals which inhibit fungal cell growth by inhibiting fun-
gal 1,3-b-D-glucan synthase.
Key words cell wall; piperazine propanol; 1,3-b-D-glucan synthase; antifungal
Antifungal agents, polyenes, and azoles are available for
Several 1,3-b-D-glucan synthase inhibitors have been iden-
the treatment of serious and life-threatening fungal infection, tified, such as echinocandins and papulacandins.19,20) Papula-
mainly caused by Candida albicans and Aspergillus fumiga- candins are liposaccharide inhibitors isolated from Papularia
tus. However, their clinical uses are restricted due to toxicity sphaerosperma. The echinocandins, including cilofungin, ac-
(polyenes), fungistatic activity (azoles), and the emergence of uleacins, and pneumocandins, are cyclic hexapeptides with a
resistant isolates (azoles).1,2) To overcome these problems, lipophilic side chain such as a linoleoyl or myristoyl moiety.
novel antifungal agents with a different mode of action are in From this chemical class, caspofungin and micafungin have
demand.
been launched recently, and anidulafungin is being devel-
1,3-b-D-Glucan synthase (UDP-glucose: 1,3-b-D-glucan 3- oped. Aerothricin3/FR901469, a cyclic lipopeptidelactone
b-D-glucosyltransferase; EC 2.4.1.34), catalyses the synthe- composed of twelve amino acids and a 3ꢀ-hydroxypalmitoyl
sis of 1,3-b-D-glucan, the most abundant component of the moiety, is another class of lipopeptide inhibitors recently iso-
fungal cell wall. 1,3-b-D-Glucan synthase is composed of lated.21—24) More recently, another class of inhibitors, terpene
a putative catalytic subunit with sixteen putative trans- glycosides, have been identified from natural sources.25,26)
membrane domains encoded by a pair of closely related
We describe here a novel piperazine propanol derivative of
genes, FKS1/GSC1/CWH53/ETG1/CND1/PBR1/YLR342W 1,3-b-D-glucan synthase inhibitor identified from a chemical
and FKS2/GSC2/G4074/YGRO32W,3—8) and a regulatory library. In vitro antifungal activity of this inhibitor was as-
subunit encoded by the RHO19,10) in Saccharomyces cere- sessed. Susceptibility analysis of S. cerevisiae fks tempera-
visiae. In C. albicans, the catalytic and regulatory subunits ture-sensitive mutants supports the conclusion that the piper-
are encoded by CaFKS1/GSC1,11,12) and CaRHO1,13) respec- azine propanol derivative inhibits fungal cell growth by in-
tively. 1,3-b-D-Glucan synthase has three features of a hibiting fungal 1,3-b-D-glucan synthase.
promising target for an antifungal agent:2,14,15) 1) its function
is essential for growth, proven by the fact that the disruption MATERIALS AND METHODS
of the genes for the catalytic subunit of 1,3-b-D-glucan syn-
thase is a lethal event in S. cerevisiae,6,8) C. albicans,11,12) and
Fungal Strains As reference strains, ATCC strains from
Cryptococcus neoformans16); 2) mammalian cells have no the American Type Culture Collection (Rockville, MD,
comparable cell wall, indicating that a 1,3-b-D-glucan syn- U.S.A.) and IFO strains from the Institute for Fermentation
thase inhibitor would be highly selective to fungal cells; and Osaka (Osaka, Japan) were purchased. Other clinical isolates
3) genes for catalytic subunits have been identified from sev- were obtained from hospitals in Japan. The S. cerevisiae
eral pathogenic fungi, such as C. albicans,11,12) Cr. neofor- strains used in this study are listed in Table 1 and were
mans,16) A. fumigatus,17) and Paracoccidioides brasiliensis,18) cultivated in YPD medium (2% Bacto peptone, 1% Bacto
and are highly homologous to each other. It is likely that 1,3- yeast extract, and 2% glucose). The YOC1071, YOC1077,
b-D-glucan synthase inhibitors might possess a broad spec- YOC1081, YOC1085, and YOC1089 have the same FKS1 al-
trum of antifungal activity.
leles as YOC1072, YOC1078, YOC1082, YOC1086, and
∗ To whom correspondence should be addressed. e-mail: kondoosm@chugai-pharm.co.jp
© 2005 Pharmaceutical Society of Japan

November 2005
2139
Table 1. S. cerevisiae Strains Used in This Study
were added. The reaction mixture was stirred at 70 °C for
1 h then concentrated under vacuum. The crude material
was purified by column chromatography (2% methanol in
dichloromethane) to afford (2,6-difluoro-phenyl)-carbamic
acid 3-(4-benzothiazol-2-yl-piperazine-1-yl)-propyl ester.
Reference/
Source
Strain
Genotype
A451
MATa ura3 leu2 trp1 can1
aro7
Refs. 6, 29
Refs. 6, 29
Refs. 6, 29
Ref. 28
b
Purification of 1,3- -D-Glucan Synthase and Measure-
Dfks1
MATa ura3 leu2 trp1 can1
aro7 gsc1::URA3
ment of Its Inhibition Membrane preparation and partial
purification of 1,3-b-D-glucan synthase were described previ-
ously.11,13) 1,3-b-D-Glucan synthase activity measurement
was performed as previously described.3) Briefly, the mem-
brane fraction was prepared from late-log phase cells and
then the enzyme was partially purified by product entrap-
ment. The partially purified enzyme was mixed with 0.1 mM
UDP-[6-3H]-glucose (222 Bq, Amersham), 75 mM Tris–Cl
pH 7.5, 0.75 mM EDTA, 25 mM KF, 20 mM guanosine-5ꢀ-
(gthio)-triphosphate, 0.1% BSA, and 7.8% Glycerol. Test
compounds were serially diluted and added. The reaction was
carried out in 100 ml at 25 °C for 30 min. After filtration and
twice washing with 70% ethanol, the radiolabeled glucose
incorporated into the polymerized glucan on the filter was
quantified by measuring the radioactivity.
Dfks2
MATa ura3 leu2 trp1 can1
aro7 gsc2::LEU2
YOC1001
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::FKS1::TRP1
YOC1095
YOC1071
YOC1077
YOC1081
YOC1085
YOC1089
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::fks1-28::TRP1
Y. Ohya
Y. Ohya
Y. Ohya
Y. Ohya
Y. Ohya
Y. Ohya
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::fks1-1014::TRP1
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::fks1-1104::TRP1
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::fks1-1125::TRP1
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::fks1-1144::TRP1
MATa ade2 his3 leu2 lys2 trp1
ura3 fks1::HIS3 fks2::LYS2
ade3::fks1-1163::TRP1
In Vitro Antifungal Activity Antifungal susceptibility
assay was performed by the NCCLS M27-A2 microdilution
method using modified media: Yeast Nitrogen Base (YNB)
supplemented with 1% glucose and 0.25% K2HPO4 was used
for the Candida spp. and, when solidified with 0.2% low-
melting temperature agarose, used for A. fumigatus. The in-
oculum size for all strains was 1 to 3ꢁ104 conidia/ml in final
concentration. 96-well plates were incubated at 35 °C for
YOC1090,27) respectively. Construction of the temperature- 1 d for C. albicans and A. fumigatus, 2 d for C. glabrata
sensitive (ts) strains and a wild-type FKS1 control YOC1001 ATCC2001, and 3 d for IFO0005. MICs were determined by
in the Dfks1 Dfks2 background28) will be described elsewhere the concentration of drug that produced an 80% reduction of
(M. Abe, M. Minemura-Asakawa, T. Utsugi, M. Sekiya- turbidity compared to that of the drug free control measured
Kawasaki, A. Hirata, H. Qadota, K. Morishita, T. Watanabe, by spectrophotometer at an optical density of 600 nm. The
and Y. Ohya, unpublished results).
susceptibility of S. cerevisiae strains was measured the same,
Chemicals The chemical library of compounds was ob- except for temperatures of 25 °C for null mutants and 25 °C,
tained from Alanex Corp. (CA, U.S.A.). Amphotericin B 31 °C, 32 °C, and 33 °C for ts mutants. IC50 values refer to
(AMB) and cycloheximide were purchased from Sigma the compound concentrations that gave 50% inhibition of
Chemical Co. (St. Louis, MO, U.S.A.). Aerothricin3 was pre- cell growth compared with the control.
pared as previously described.29)
GSI578 [(2,6-Difluoro-phenyl)-carbamic acid 3-(4-ben- RESULTS AND DISCUSSION
zothiazol-2-yl-piperazine-1-yl)-propyl ester] was synthesized
as follows. To a solution of 2-chloro-benzothiazole in
In order to identify novel chemical classes of antifungals,
toluene, piperazine and diisopropylethylamine were added. screening of a chemical library was conducted by monitoring
The reaction mixture was stirred at 120 °C for 18 h, then the inhibition of 1,3-b-D-glucan synthase activity. 1,3-b-D-
added water and extracted with dichloromethane. The Glucan synthase was partially purified by product entrapment
organic phase was dried with magnesium sulfate and from the pathogenic fungus C. albicans IFO1060.11,13) We
concentrated under vacuum. Recrystallization from identified GSI578 [(2,6-Difluoro-phenyl)-carbamic acid 3-(4-
dichloromethane and hexane gave 2-piperazine-1-yl-benzo- benzothiazol-2-yl-piperazine-1-yl)-propyl ester] as a potent
thiazole. To a solution of 2-piperazine-1-yl-benzothiazole 1,3-b-D-glucan synthase inhibitor (Fig. 1). The IC50 value
in toluene, 3-bromo-propan-1-ol and diisopropylethylamine of GSI578 against 1,3-b-D-glucan synthase was 0.16 mM,
were added. The reaction mixture was stirred at 120 °C for whereas that of aerothricin3 was 0.012 mM.
2 h, then added water and extracted with dichloromethane.
The inhibitory activity of GSI578 against the growth
The organic phase was dried with magnesium sulfate and of various fungal strains was compared to caspofungin,
concentrated under vacuum. The crude material was purified aerothricin3, and amphotericin B (Table 2). GSI578 exhib-
by column chromatography with 2—5% methanol in ited antifungal activity against C. albicans, C. glabrata, and
dichloromethane and recrystallized from dichloromethane A. fumigatus comparative to the reference antifungals.
and hexane to afford 3-(4-benzo-thiazol-2-yl-piperazine-1-
To explore the inhibition of 1,3-b-D-glucan synthesis by
yl)-propan-1-ol. To a solution of 3-(4-benzo-thiazol-2-yl- the piperazine propanol derivative, we measured the suscepti-
piperazine-1-yl)-propan-1-ol in tetrahydrofuran, 1,3-difluoro- bility of fks1 ts mutants in the Dfks2 background at semiper-
2-isocyanato-benzene and catalytic amount of pyridine missive and permissive temperatures. Figure 2 shows that ts

2140
Vol. 28, No. 11
Table 3. Susceptibility of S. cerevisiae fks Null Mutants
Growth inhibition: IC50 [mg/ml]
Strain
GSI578
Aerothricin3
Cycloheximide
Fig. 1. The Chemical Structure of GSI578
Dfks1
Dfks2
A451
0.50ꢂ0.006
0.36ꢂ0.011
0.35ꢂ0.007
0.0033ꢂ0.001
0.16ꢂ0.004
0.12ꢂ0.005
0.027ꢂ0.001
0.028ꢂ0.002
0.030ꢂ0.001
Table 2. Susceptibility of Pathogenic Fungi to GSI578
MIC [mg/ml]
GSI578 Aerothricin3 Caspofungin AMB
The yeast growth was measured by absorbance at 600 nm. Values are the averages of
three independent experiments and standard deviations.
Strain
strain (Table 3) as well as the control drug cycloheximide.
These results considered with the abovementioned results on
the ts mutants, suggest that the mechanism of 1,3-b-D-glucan
synthase inhibition by GSI578 is different from that of cyclic
lipopeptide inhibitors such as aerothricin3 and caspofungin.
We here identified a novel chemical class of 1,3-b-D-glu-
can synthase inhibitors which exhibits potent in vitro antifun-
gal activity. Susceptibility analysis of the fks mutant strains
suggested that the piperazine propanol derivative inhibits
1,3-b-D-glucan synthase by an action mode different from
known 1,3-b-D-glucan synthase inhibitors. Therefore, the
piperazine propanol derivative might achieve 1,3-b-D-glucan
synthase inhibition in strains resistant to known 1,3-b-D-glu-
can synthase inhibitors. Also, complete inhibition of 1,3-b-D-
glucan synthase might be possible with our piperazine
propanol derivative in combination with known 1,3-b-D-glu-
can synthase inhibitors.
C. albicans ATCC48130 0.26
0.0078
0.056
0.044
0.0081
0.39
0.70
0.44
0.19
0.33
0.18
0.32
0.36
0.34
0.52
0.72
0.35
0.27
0.32
0.044
0.089
0.19
0.088
0.35
0.35
0.29
0.22
0.28
CY3003
CY1123
0.83
1.3
CY1124
C. glabrata ATCC2001
IFO0005
A. fumigatus CF1083
CF1003
0.71
0.17
0.19
1.1
0.15
0.47
CF924390
The growth of fungi was measured by absorbance at 600 nm.
The piperazine propanol GSI578 represents the first non-
natural product inhibitor of 1,3-b-D-glucan synthase. With
pharmacological assessment and chemical modification of
the piperazine propanol derivative, therapies for fungal infec-
tions may possibly be expanded.
Acknowledgements This work was supported in part by
International Research Grant K-1023 from the Japan Health
Sciences Foundation. We are grateful to F. Ford for proof-
reading this manuscript.
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