Bioassay-guided isolation of novel and selective urease inhibitors from Diospyros lotus / 中国天然药物

Two new dimeric naphthoquinones, 5',8'-dihydroxy-6,6'-dimethyl-7,3'-binaphthyl-1,4,1',4'-tetraone (1; Di-naphthodiospyrol D) and 5',8'-dihydroxy-5,8-dimethoxy-6,6'-dimethyl-7,3'-binaphthyl-1,4,1',4'-tetraone (2; Di-naphthodiospyrol E), along with known naphthoquinones diospyrin (3) and 8-hydroxy diospyrin (4) were isolated from the chloroform fraction of extract of Diospyros lotus roots. Their structures were elucidated by advanced spectroscopic analyses, including HSQC, HMBC, NOESY, and J-resolved NMR experiments. The fractions and compounds 1-4 were evaluated for urease activity and phosphodiesterase-I, carbonic anhydrase-II and α-chymotrypsin enzyme inhibitory activities. Compounds 1 and 2 and their corresponding fractions showed significant and selective inhibitory effects on urease activities. The IC values of 1 and 2 were 260.4 ± 6.37 and 381.4 ± 4.80 µmol·L, respectively, using thiourea (IC = 21 ± 0.11 µmol·L) as the standard inhibitor. This was the first report demonstrating that the naphthoquinones class showed urease inhibition.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Biochemical characterization and some biological properties of the phosphodiesterase I purified from Agistrodon bilineatus venom.

The venom phosphodiesterase I (PDE-I, EC 3.1.4.1) is useful in the elucidation of the structure and nucleotide sequence of nucleic acids. In the present study, PDE-I was purified from Agistrodon bilineatus venom by preparative native-PAGE. A single protein band was observed in analytical native-PAGE. The enzyme also gave a single band in SDS-PAGE with a molecular mass of 140 kDa. The position of the band was not altered in the presence of β-mercaptoethanol, suggesting the protein did not contain subunits. The enzyme was free from 5’-nucleotidase and alkaline phosphatase activities. It showed a broad optimum pH range (9.0-11.0), whereas the optimum temperature was found to be 600C, with activity decreasing at >650C. Energy of activation (Ea) was calculated to be 0.31. The PDE-I was a glycoprotein having 14% of carbohydrate content. The Vmax, Km, Kcat and Ksp values of the enzyme were 3.85 μM/min/mg, 8.3 × 10-3 M, 23s-1 and 46.4 M-1 Min-1 respectively. Cysteine caused a non-competitive inhibition with a Ki 6.3 × 10−3 M (IC50 of 1.6 mM), whereas ADP caused a competitive inhibition having Ki 0.8 × 10−3 M (IC50 5.4 mM). Glutathione, o-phenanthroline, zinc and EDTA inhibited the enzyme activity, whereas Mg2+ slightly potentiated the activity. The enzyme hydrolyzed thymidine 5’-monophosphate p-nitro-phenyl ester most readily (10-fold), while 3’-5’-cAMP was least readily hydrolyzed substrate. The enzyme up to 4.0 mg/Kg i.p was not lethal in mice. It exhibited an anticoagulant effect, and increased the normal clotting time of normal citrated human plasma, whereas the crude venom showed strong coagulant effect. The above results showed that the A. bilineatus PDE-I was very similar to that isolated from other snake venoms. The purification procedure described here is simple, rapid and reproducible and may prove useful to isolate pure protein for investigation into the contribution of this enzyme to the biological activities of A. bilineatus venom and PDE-I insight, in general.