全 文 ：组织蛋白酶 B 的新型天然抑制剂 ?
贾锐锐 , 曾广智 , 谭宁华?? , 周志宏 , 张颖君
(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 : 组织蛋白酶 B 是木瓜蛋白酶类半胱氨酸蛋白酶家族的重要成员 , 它与人类多种疾病相关 , 尤其是在
恶性肿瘤的侵袭转移过程中扮演了重要角色。通过随机筛选 , 发现了五个对组织蛋白酶 B 具有较好抑制活
性的天然化合物 prodelphinidin B-2 3′-O-gallate (1) , prodelphinidin B-2 (2) , procyanidinB-2 (3) , puerin A (4) 和
( -) epigallocatechin-3-O-gallate (5) , 其 IC50 值分别为 0.58 , 0 .44 , 0 .76 , 2 .07 和 0 .96μmol?L。这五个抑制剂为
黄烷醇类化合物 , 均为组织蛋白酶 B 的新型天然抑制剂。
关键词 : 组织蛋白酶 B; 天然抑制剂 ; 黄烷醇
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 05 - 621 - 03
New Natural Inhibitors of Human Cathepsin B *
J IA Rui-Rui , ZENG Guang-Zhi , TAN Ning-Hua
* *
, ZHOU Zhi-Hong, ZHANG Ying-Jun
( State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204 , China)
Abstract : Cathepsin B isan important member of thepapain-likecysteineproteases . It has been implicated in the patholo-
gy of many human diseases, especially in the invading and metastasis of most malignant tumors . With randomscreening,
five natural compounds, prodelphinidin B-2 3′-O-gallate (1 ) , prodelphinidin B-2 ( 2) , procyanidin B-2 ( 3) , puerin A
(4) and (- ) epigallocatechin-3-O-gallate ( 5 ) , were found to show potent cathepsin B inhibitory activity with IC50 s of
0 .58 , 0 .44 , 0 .76 , 2 .07 and0 .96μmol?L , respectively . Inhibitors 1 - 5 arefoundwith chemotype of flavanols, which are
new natural inhibitors of cathepsin B .
Key words: Cathepsin B ; Natural inhibitors; Flavanols
Cathepsins, the main member of the papain-like
cysteine proteases, comprise agroupof proteolytic enz-
ymes involved in many human physiological processes,
and theoverexpressionof these enzymeswill lead tose-
vere diseases ( Lecaille et al. , 2002; Turk et al. ,
2000) .
Cathepsin B ( CatB) is oneof themost extensively
investigated members of the papain-like cysteine pro-
teases . It has been involved invarious humandiseases,
such as tumor, osteoporosis, rheumatoid arthritis and
parasitic diseases etc (Lecaille et al. , 2002; Otto and
Schirmeister, 1997; Zeng et al. , 2005 ) . Recently,
the important roleof CatB in tumor metastasis has been
well documented, whichdemonstrated thatCatB can en-
hance theinvasion of many tumors by aiding in extracel-
lular matrix breakdown . Moreover, increased expression
and?or activityof CatB havebeenobserved inmanymet-
astatic tumors . It suggested that CatB is implicated in
the development, invasion and metastasis of tumors .
Therefore, CatB is considered as an important target in
云 南 植 物 研 究 2008 , 30 (5) : 621～623
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07305
?
?? ?Author for correspondence; Tel?Fax: 0871 - 5223800; E-mail : nhtan@ mail .kib. ac. cn
Received date: 2007 - 12 - 20 , Accepted date: 2008 - 01 - 03
作者简介 : 贾锐锐 (1974 - ) 女 , 硕士 , 主要从事体外抗肿瘤活性天然产物研究。 ?
Foun ?dation items: The National Natural Science Foundation of China (30725048) and the Foundation of ChineseAcademy of Sciences ( West Light
Program)
cancer intervention, and its inhibitors are highly sought
as potential anticancer and antimetastatic agents ( Le-
caille et al. , 2002; Turk et al. , 2000; Zeng et al. ,
2005; Lim et al. , 2004; Koblinski et al. , 2000; Ber-
dowska, 2004) .
Many anticancer drugs have originated from natu-
ral products ( Tan et al. , 2006 ) . In our previous
work, six biflavones isolated from gymnosperm plant
and one carbazole alkaloid isolated from Murraya koe-
nigii were found to benovel natural inhibitors of human
cathepsin B ( Zhang et al. , 2005; Zeng et al. , 2006;
Wang et al. , 2003) . Thestructure-activity relationship
between these six biflavones and CatB was further ana-
lyzed (Zeng et al. , 2006; Pan et al. , 2005 ) .The fol-
lowing screening results of CatB assay indicated that
five natural flavanols ( 1 - 5 ) , isolated from various
plant resources, were found to show potent inhibitory
activity against human cathepsin B (Fig . 1) .
Fig . 1 Chemical structures of compounds 1 - 5
Materials and Methods
Inhibitors 1 - 5 were isolated from plants ( Table 1 ) , and
detailed purification and identification of these compounds were
described by us previously (Zhouand Yang, 2000; Zhou et al. ,
2005; Zhou et al. , 2000) . Purities of these compoundswere >
95% . Inhibitory activities against human cathepsin B ( Cat #
219364 , Calbiochem) were determined spectrofluorometrically
according to the method we used before ( Zeng et al. , 2006 ) .
Leupeptin ( Cat # L9783 , Sigma) , a well known inhibitor for
cathepsins, was used as reference compound in this assay . The
IC50 values were calculated by dose-response curves with the
highest tested concentration of 2 .5μg?mL .
Results and Discussion
Results from CatB inhibition assay showed that
compound 1 - 3 , three biflavanols, showed potent CatB
inhibitory activities with IC50 values of 0 .58, 0.44 and
0.76μmol?L , respectively (Table 1) , which indicated
that the number of hydroxyl substitutionmay be has effect
on the activity of this kindof inhibitors . Interestingly,
Table 1 Inhibitory activity of compounds 1 - 5
against human cathepsin B
Comp . Sources IC50 ?(μmol?L )
1 hMyrica nana 0 c. 58±0 I. 21
2 hM G. nana 0 c. 44±0 I. 15
3 hCamellia saluenensis 0 c. 76±0 I. 16
4 hC ;. sinensis var ?. assamica 2 c. 07±0 I. 05
5 hMyrica nana 0 c. 96±0 I. 09
Leupeptin ——— 0 c. 05±0 I. 01
226 云 南 植 物 研 究 30 卷
most of the natural CatB inhibitors we have found are
with dimeric structures, such as alkaloid (Wang et al . ,
2003 ) , biflavone ( Pan et al . , 2005; Zeng et al . ,
2006) and biflavanol (1 - 3) .
Tumor cell invasion involves attachment of tumor
cells to the underlying basement membrane, local pro-
teolysis andmigrationof tumor cells through theproteo-
lytically modified region ( Koblinski et al. , 2000 ) .
CatB plays an important role in local proteolysis by de-
grading extracellular matrix and basement membrane,
and enhances tumor invasion and metastasis (Koblinski
et al. , 2000; Berdowska, 2004; Turk et al. , 2000;
Zeng et al. , 2005 ) . CatB overexpression has been
demonstrated in many human tumors including breast,
lung, brain, gastrointestinal , head and neck cancer,
and melanoma ( Koblinski et al. , 2000; Berdowska,
2004; Sloane et al. , 1981) . In vitro and in vivostud-
ies have shown that protease inhibitors can reduce the
invasive and metastatic capabilities of tumor cells
(Koblinski et al. , 2000 ) . Several types of chemical
functionality have served as the central pharmacophore
for cysteine proteases inhibitors ( Lecaille et al. ,
2002) . However, most of inhibitors are peptide-based
molecules . In this work, five flavanols were found as
novel natural inhibitors of CatB, which provided us the
new insight into the research of CatB inhibitors .
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3265 期 J IA Rui-Rui et al . : New Natural Inhibitors of Human Cathepsin B