The Structure Activity Relationship And Target Identification Of Erianin, An Active Compound Of D. Chrysotoxum Lindl

Objective Erianin, originally isolated from traditional Chinese medicine D. Chrysotoxum Lindl, is a kind of biphenyl natural product of low molecular weight. Emerging research has shown that erianin potently inhibits proliferation and promotes apoptosis of cancer cells such as colon, bladder, liver, gastric and melanoma, but the structure-activity relationship and target of erianin remains unknown. In this study, the preliminary structure-activity relationship of erianin and identification of its target was further investigated.Methods Firstly, six analogues of erianin that were modified by hydroxyl ethoxy, hydroxyl and hydroxyethyl respectively were designed and synthesized. The viability of HepG2 cells treated by analogues was evaluated by MTT assay to illuminate its preliminary structure-activity relationship and screen the active and inactive analogue. For PAL-ABPP strategy, seven active PAL-ABPP probes were designed and synthesized, and the viability of HepG2 cells treated by probes was evaluated by MTT assay to screen the active probe. The probes were pull down with HepG2 total protein to assay the specific protein for testing whether the PAL-ABPP method could be applied to target identification of erianin. For PAL-CC-ABPP strategy, three active probes, an inactive and a blank probe was designed and synthesized. The viability of HepG2 cells treated by the probes was evaluated by MTT assay to screen the active probe for testing whether the PAL-CC-ABPP strategy could be applied to target identification of erianin. The specific band between the active and inactive probe was investigated by pull down, silver staining and Western blot, and identified by MS.Results First of all, the six erianin analogues, seven active PAL-ABPP probes and five PAL-CC-ABPP probes were achieved by chemical synthesis and their structures were confirmed by 1H NMR and mass spectrum. Secondly, the results in vitro indicated that the antiproliferative activity of analogues 1-4 that benzene ring was modified by hydroxyethoxy was conspicuously weaker than erianin, but the antiproliferative activity of 5 and 6 that were modified by hydroxyl and hydroxyethyl respectively was basically equivalent to erianin. Moreover, seven PAL-ABPP probes that L-lysine and analogue 6 served as skeleton and active group respectively were inactive and the results of pull down indicated that any specific bands was presentd in gel by silver staining. In three PAL-CC-ABPP probes which were introduced the analogue 6 as active group, only the probe that ε-NH2 of N3-a-L-Lysine was introduced by diazirine was active except the rest of probes including the inactive and blank probe. At last, the results of pull down and silver staining indicated that a conspicuous specific band presented in active probe between 100 and 150 kD. That the biotin labeled protein at the same location was tested by Western blot further illustrated the specific protein was captured and labeled by active probe. The results of MS indicated that the specific protein was a kind of metabolic enzyme of mitochondria.Conclusions Fristly, C-7 in erianin can be used as an important modification site, and the functional groups in A and B rings are active essential, and three-dimensional conformation of AB ring is also important. Secondly, the PAL-ABPP method is not applicable to target identification of erianin. Thirdly, the activity of PAL-CC-ABPP probe 16 is not determined by N3-a-L-Lysine or the introduction of photocrosslinking group diazirine but its active group, and the PAL-CC-ABPP strategy is appropriate for target identification of erianin. Last but not least, the specific protein, labeled by active probe via the PAL-CC-ABPP strategy and a kind of metabolic enzyme of mitochondria identified by MS, might be the target of erianin. The research further lays the foundation of in-depth structure-activity relationship, subsequent target verification and illustration of intensive anti-cancer mechanism of erianin. The PAL-CC-ABPP method that N3-a-L-Lysine and diazirine serves as the skeleton and the photocrosslinking group respectively opened up for the frist time shows a promising future in target identification of small molecule drugs.