Fig. 3 Dixon plots of inhibition. Left: inhibition of
BACE1 by hispidin. Right: inhibition of PEP by hispidin.
Substrate concentration: Left; ±!± 250 nM, ±*± 500
nM, ±l± 750 nM. Right; ±!± 0.5 mM, ±*± 0.75 mM,
±l± 1.0 mM.
7 Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan Met al.
Purification and cloning of amyloid precursor protein b-secretase
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Table 1 Inhibitory activitiesa of hispidin against TACE, PEP, and other
serine proteases
8 Vassar R, Bennett BD, Babu-Khan S, Khan S, Mendiaz A, Denis Pet al.
b-Secretase cleavage of Alzheimer's amyloid precursor protein by the
transmembrane aspartic protease BACE1. Science 1999; 286: 735±41
9 Schmidt B. Aspartic protease involved in Alzheimer's disease. Chem-
BioChem 2003; 4: 366±78
Conc.
ꢀmM)
Chymo- Trypsin
trypsin
Elastase PEP
TACE
BACE1
10
Hispidin
4.0
Ghosh AK, Shin D, Downs B, Koelsch G, Lin X, Ermolieff JJ et al. Design
of potent inhibitors for human brain memapsin 2 ꢀb-secretase). J Am
Chem Soc 2000; 122: 3522±3
Bakker AV, Jung S, Spencer RW, Vinick FJ, Faraci WS. Slow-tight bind-
ing of prolyl endopeptidase by benzyloxycarbonylprolinal. Biochem J
1990; 274: 559±62
Toda S, Obi Y, Kimura K, Hamagishi Y, Tomita K, Komiyana Net al. Eury-
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production, isolation and biological activities. J. Antibiotics 1992; 45:
1573±9
0.6 0.2 3.3 0.3 5.2 1.2 30.1 1.2 0.1 0.0 53.0 0.8
1.4 0.3 1.0 0.2 3.1 1.3 61.3 7.0 1.1 0.1 63.4 3.1
0.6 0.2 0.0 0.0 8.2 2.0 80.1 5.8 3.1 1.0 81.1 4.9
4.4 1.3 1.6 0.2 8.1 0.2 3.1 3.2 2.1 0.8 10.1 0.8
20.0
11
12
40.0
Controlb
EGCGc
ZPPCHOc
±
±
±
±
±
±
±
±
±
81.2 5.0
±
50.0 5.6
a The activities %%) are mean SE of three independent experiments. The inhibition ratio was
13
14
15
Chung CH, Ines HE, Almeda S, Goldberg AL. Purification of Escherichia
coli of a periplasmic protein that is potent inhibitor of pancreatic pro-
tease. J Biol Chem 1983; 258: 11032±8
Hubert F, Gaertner J, Antonie J, Puigserver AJ. Increased activity and
stability of polyethyleneglycol-modified trypsin. Enzyme Microb
Technol 1992; 14: 150±5
Bieth J, Spiess G, Camille GW. The synthesis and analytical use of a
highly sensitive and convenient substrate of elastase. Biochem Med
1974; 11: 350±7
Adam W, Saha-Moeller CR, Veit M, Markus W, Welke B. A convenient
synthesis of hispidin from piperonal. Synthesis, 1994: 1133±4
Klaar M, Wolfgang S. Fungus pigments, XXVII. Isolation of hispidin
and 3,14¢-bihispidinyl from Phellinus pomaceus ꢀPoriales). Chem Ber
1997; 110: 1058±62
Gonindard C, Bergonzi C, Denier C, Sergheraert C, Klaebe A, Chavant
Let al. Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward
cancer cells than normal cells in vitro. Cell Biol Toxicol 1997; 13:
141±53
Buxbaum JD, Liu K-N, Luo Y, Slack JL, Stocking KL, Peschon JJet al. Evi-
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precursor. J Biol Chem 1998; 273: 27765±7
calculated as described in Materials and Methods.
b Ten mL of 5% MeOH were added to the reaction mixture instead of a sample solution.
Eleven mM of %±)-epigallocatechin gallate and 7 nM of Z-Pro-prolinal were used as positive
controls for BACE1 and PEP, respectively.
c
146
secure activity [23]. The Ki and IC50 values of hispidin were much
higher than those of synthetic peptidomimetics, but relatively
similar to those of a natural inhibitor EGCG ꢀIC50 = 1.610±6 M,
Ki = 2.110±7 M) [24], which was introduced as a positive con-
trol for BACE1 inhibition in this study. Enzyme inhibitors with
therapeutic potential are preferably smaller than 700 Da with
an IC50 value in the nM level, so hispidin might not be directly
considered as a drug candidate. However, it might be a starting
point for rational non-peptidyl drug design and be a useful re-
agent for studying the enzyme properties of BACE1.
16
17
18
19
20 Katsube N, Sunaga K, Chuang D-M, Ishitani R. ONO-1603, a potential
antidementia drug, shows neuroprotective effects and increases m3-
muscarinic receptor mRNA levels in differentiating rat cerebellar
granule neurons. Neurosci Lett 1996; 214: 151±4
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