Natural product juglone targets three key enzymes from Helicobacter pylori: inhibition assay with crystal structure characterization.
OBJECTIVE
To investigate the inhibition features of the natural product juglone (5- hydroxy-1,4-naphthoquinone) against the three key enzymes from Helicobacter pylori (cystathionine gamma-synthase [HpCGS], malonyl-CoA:acyl carrier protein transacylase [HpFabD], and beta-hydroxyacyl-ACP dehydratase [HpFabZ]).


METHODS
An enzyme inhibition assay against HpCGS was carried out by using a continuous coupled spectrophotometric assay approach. The inhibition assay of HpFabD was performed based on the alpha-ketoglutarate dehydrogenase-coupled system, while the inhibition assay for HpFabZ was monitored by detecting the decrease in absorbance at 260 nm with crotonoyl-CoA conversion to beta -hydroxybutyryl-CoA. The juglone/FabZ complex crystal was obtained by soaking juglone into the HpFabZ crystal, and the X-ray crystal structure of the complex was analyzed by molecular replacement approach.


RESULTS
Juglone was shown to potently inhibit HpCGS, HpFabD, and HpFabZ with the half maximal inhibitory concentration IC50 values of 7.0 +/-0.7, 20 +/-1, and 30 +/-4 micromol/L, respectively. An inhibition-type study indicated that juglone was a non-competitive inhibitor of HpCGS against O-succinyl- L-homoserine (Ki=alphaKi=24 micromol/L), an uncompetitive inhibitor of HpFabD against malonyl-CoA (alphaKi=7.4 micromol/L), and a competitive inhibitor of HpFabZ against crotonoyl-CoA (Ki=6.8 micromol/L). Moreover, the crystal structure of the HpFabZ/juglone complex further revealed the essential binding pattern of juglone against HpFabZ at the atomic level.


CONCLUSIONS
HpCGS, HpFabD, and HpFabZ are potential targets of juglone.
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yun-hua
YH
Kong
Liang
L
Zhang
Zheng-yi
ZY
Yang
Cong
C
Han
Li-hong
LH
Hu
Hua-liang
HL
Jiang
Xu
X
Shen
D003460:Crystallization; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016480:Helicobacter pylori / Q000187:drug effects / Q000201:enzymology*; D009285:Naphthoquinones / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D014961:X-Ray Diffraction
D004791:Enzyme Inhibitors; D009285:Naphthoquinones; C005134:juglone