Abstract
A novel in vitro strategy for affinity-based ultrafiltration-high performance liquid chromatography (HPLC) was developed for the direct identification of cytochrome P450 (CYP) 1A2, 3A4, and 2C9 specific ligands from Danshen extracts, in which human liver microsome (HLM) was used as the source of CYP enzymes. The Danshen extract was incubated without HLM, with HLM, or with HLM where the active site of the target enzyme was blocked with a specific competitive probe before ultrafiltration to isolate ligand–enzyme complexes from unbound compounds. Subsequently, HPLC analysis was performed on the released ligands from the complexes. α-Naphthoflavone, ketoconazole, and sulfaphenazole were used as specific competitive probes for CYP1A2, 3A4, and 2C9, respectively. The signal-to-noise ratio (S/N) and specific-signal-to-noise ratio (S-S/N) of each compound were calculated using the obtained peak areas. Finally, two criteria were applied to select putative ligands for each specific target: (1) S/N > 1; (2) S-S/N > 0. Finally, dihydrotanshinone was identified as a specific ligand for CYP1A2 and tanshinone I, cryptotanshinone, and tanshinone IIA were identified as specific ligands for CYP1A2, 2C9, and 3A4. It was demonstrated that the newly developed method can be used to rapidly and directly detect specific ligands from natural product extracts in multi-target systems.
Highlights
Dihydrotanshinone was identified as a specific ligand for CYP1A2 and tanshinone I, cryptotanshinone, and tanshinone IIA were identified as specific ligands for CYP1A2, 2C9, and 3A4
The major principles are as follows: a solution of the natural product matrix such as Danshen (Salvia miltiorrhiza) ethanol extracts (DEE) is incubated with the target mixture (i.e., human liver microsome (HLM))
The results revealed that these tanshinones are ligands of CYP1A2, 3A4 and 2C9
Summary
Affinity-based ultra ltration-high performance liquid chromatography (HPLC) has emerged as a rapid and convenient approach for protein–ligand interaction studies, and has been widely applied for the screening and identi cation of ligands of enzymes, proteins, or receptors from natural product matrices, in which small molecules from natural product extracts bound to selected targets (enzymes, proteins, or receptors) can be separated from unbound small molecules by ultra ltration and subsequently identi ed.[5] examination of the HPLC pro le of certain peaks can be used to identify “hits”. Affinity-based ultra ltration-HPLC has signi cant advantages including rapid and convenient operation, low cost, high hit ratio, and lack of immobilization of screening targets on a carrier as in other active molecular screening methods such as bioassay-. Previous studies have only described multi-ligand screening in single-target systems on affinity-based ultra ltration-HPLC platforms, and using this method for screening in a multi-target system is still a challenge
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