全 文 ：以细菌中烯醇式丙酮酸转移酶为作用
靶点的新天然抑制剂?
姜立花 , 谭宁华 ** , 曾广智?? , 郝小江 , 张颖君 , 邱明华
(中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室 , 云南 昆明 650204 )
摘要 : 肽聚糖是细菌细胞壁的重要成分 , 烯醇式丙酮酸转移酶 (EPT) 是调节肽聚糖合成初始阶段的关键
酶。通过活性筛选 , 发现了三个对 EPT有抑制活性的天然产物 (化合物 1～3) , 它们皆为 EPT 的新抑制
剂。体外抗真菌实验发现 , 化合物 2 和 3 对光滑念珠菌具有较好的生长抑制作用。另外 , 化合物 3 还具有
一定的肿瘤细胞毒活性。
关键词 : 烯醇式丙酮酸转移酶 ; 天然抑制剂 ; 细胞壁 ; 抗菌
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 03 - 378 - 03
New Natural Inhibitors Targeting Bacterial
Enolpyruvyl Transferase *
J IANG Li-Hua, TAN Ning-Hua** , ZENG Guang-Zhi ** ,
HAO Xiao-Jiang, ZHANG Ying-Jun, QIU Ming-Hua
( State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204 , China)
Abstract: Bacterial enolpyruvyl transferase (EPT ) is one of the key enzymes acting in the initial stage of peptidoglycan
synthesis of bacterial cell walls . Through inhibitionassay, threenewnatural inhibitors (1 - 3) were found against EPT . In-
hibitor 2 and 3 showed antifungal activities against fungus Candida glabrata, and inhibitor 3 showed cytotoxicities against
several cancer cells .
Key words: Enolpyruvyl transferase; Natural inhibitor; Cell wall ; Antibacterial
Bacterial cells possess a cell wall , while myco-
plasma or mammalian cells don′t possess it . Pepti-
doglycan is the major structural component of bacterial
cell walls . Inhibition of its biosynthesis will prevent
cell wall formation and lead to cell death eventually .
Thus, peptidoglycan is an important target of many
clinically used antibiotics, such as penicillin, β-lac-
tams, imipenems, cephalosporins and glycopeptides
(Yuan et al. , 2007) .
Peptidoglycan is aheteropolymer comprisedof lin-
ear chains of polysaccharides containing N-acetylmu-
ramic acid and N-acetylglucosamine, cross-linked by
short peptidic chains ( Lortal and Chapot-Chartier,
2005) . Many enzymes involved in peptidoglycan bio-
synthesis, such as N-acetyl-glucosamine-1-phosphate
uridyltransferase (GlmU) (Sulzenbacher et al. , 2001) ,
UDP-N-acetyl-glucosamine enolpyruvyl transferase ( EPT
or MurA ) ( Bachelier et al. , 2006 ) , UDP-N-acetyl-
云 南 植 物 研 究 2008 , 30 (3) : 378～380
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07289
?
?? ?Author for correspondence; Tel?Fax: 86 - 871 - 5223800; E-mail : nhtan@ mail . kib. ac. cn, gzh—zeng@ mail . kib. ac. cn
Received date: 2007 - 12 - 12 , Accepted date: 2008 - 01 - 15
作者简介 : 姜立花 (1975 - ) 女 , 硕士 , 主要从事生物活性筛选研究。 ?
Foundation ?item: TheNational Natural ScienceFoundation of China ( 30725048) , theFoundation of ChineseAcademy of Sciences ( WestLight Pro-
gram)
enolpyruvyl glucosamine reductase (MurB) ( Benson et
al. , 1996) , penicillin-binding proteins (PBPs) (Tuo-
manen, 1986 ) and D-glutamic acid-adding enzyme
(MurD ) (?trancar et al. , 2006 ) etc . EPT ( EC
2 .5 .1 .7) is responsible for the first step in the cyto-
plasmatic biosynthesis of peptidoglycan precursor mole-
cules . It catalyzes the transfer of phosphoenolpyruvate
(PEP) to the 3-hydroxyl group of UDP-N-acetylglu-
cosamine ( UDPAG ) yielding enolpyruvyl-UDP-N-
acetylglucosamine and inorganic phosphate ( Bachelier
et al. , 2006 ) . For its potential pharmaceutical interest
in antibacterial agents, EPT was an attractive target for
antibiotic discovery . Many EPT specific inhibitors were
found, such as fosfomycin, derivatives of 5-sulfonoxy-
anthranilic acid, pyrazolopyrimidine and cyclic disul-
fide (Baum et al. , 2001; Eschenburg et al. , 2005 ) ,
and some of them showed good inhibitory activities
against bacteria .
In our previous research for natural antibacterial
inhibitors, some extracts from plants were found with
potent inhibitory activities against EPT ( J iang et al. ,
2003) . In this paper, three new natural inhibitors ( 1
- 3 ) of EPT were found with IC50 s below 50 μg?mL
(Fig . 1) through EPT assay . The antifungal results in-
dicated that compounds2 and 3 showed inhibitoryactivi-
ties against fungus Candida glabrata (CG) (Table 1) .
Materials and Methods
Compounds 1 - 3 (Fig . 1) were isolated by us with purities
> 95% . Detailed purifications and identificationswere described
before (Zhou et al. , 2005; Stermitz and Castro, 1983; Mu and
Fig . 1 Chemical structures of natural inhibitors 1 - 3
Li , 1982) . Fosfomycin (Sigma, P5396 ) is used as a reference
compound for EPT assay .
Inhibitory activity against EPT was performed as described
previously ( Baum et al. , 2001 ; Eschenburg et al. , 2005 ) .
Compounds were diluted with reaction buffer ( pH 7.4 , 12 .5
μmol?L Tris-HCl , 5% BSA-Tris) in the presenceof 1μg?mL En-
terobacter cloacaeEPT solution ( provided by Bayer AG (Germa-
ny) ) . Then the reaction was initiated by the addition of 125
μmol?L UDPAG (Sigma, U-4375) and 32 .5μmol?L PEP (Flu-
ka, 79415) . After 2 h functional time at 37℃ , indicator con-
taining 0 .045% malachite green base and 3 .16% ammonium
molybdate tetrahydrate was added, and the OD values at 630 nm
were measured in a plate reader (Molecular Devices, Spectra
MAX340) . IC50 s of compounds against fungus CG were deter-
mined by Yeast Nitrogen Glucose ( YNG) method . Test com-
pounds were diluted with the YNG medium ( 14.5 mg?mL
Na2 HPO4·12H2 O, 3 . 55 mg?mL KH2 PO4 , 6 . 7 mg?mL yeast ni-
trogen base dehydrated, 10 mg?mL glucose) . Inocula containing
1 .3×103 cells?mL of CG in log phase growth were prepared in
YNG cultures . After incubated for 55 h at 37℃ , the OD values
weremonitored at 540 nmin a plate reader (Molecular Devices,
spectra MAX340) .
Results and Discussion
The increasing number of antibiotic-resistant bac-
teria has fuelled interest in the development of new an-
tibiotics and other antibacterial agents . EPT , one of
the important enzymes of peptidoglycan biosynthesis of
bacterial cell walls, is apotential target for antibacteri-
al agents ( Lanzetta et al. , 1979 ) . Through inhibition
assay, three new natural inhibitors of EPT were found
with chemotypes of flavone (1 ) , flavane ( 2) and alka-
loid (3) ( Fig . 1) . These three inhibitors have no ap-
parently structural similarity to fosfomycin . Moreover,
the antifungal tests also showed that two inhibitors of
them ( 2 and 3 ) are with potent inhibitory activities
against fungus CG ( Table 1 ) , which indicated that
EPT inhibition might be one of their antibacterial
Table 1 Inhibitory activities of inhibitors 1 - 3
against bacterial EPT and fungus CG
Comp . Plant sources
IC50 ?(μg?mL )
EPT CG-YNG
1 ?Artocarpus pithecoqallus 33 .29 NA
2 ?Camellia sinensis var =. assamica 41 .35 2 ?. 88
3 ?Michelia yunnanensis 40 .47 3 ?. 90
NA , Not Active
9733 期 JIANG Li-Hua et al .: New Natural Inhibitors Targeting Bacterial Enolpyruvyl Transferase
mechanisms . Additionally, compound 1 - 3 were tested
for their cytotoxicities against cancer cells by sulfurho-
damine B assay (Skehan et al. , 1990 ) . Results indi-
cated that compound 3 showed cytotoxicities against
cancer cell line A549 ( lung cancer) , BGC-823 ( gas-
tric cancer cell line) and MDA-MB-231 ( breast cancer
cell line) with IC50 s below 10μg?mL .
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