Abstract
Parkin is a crucial E3 ubiquitin ligase for initiating mitophagy through the PINK1/Parkin pathway. Regulating the expression and activity of parkin can remedy mitophagy and human disease. We developed an efficient method to isolate natural parkin ligands from herbal medicines by combining centrifugal ultrafiltration and liquid chromatography/mass spectrometry. The heterologous expression technology identified functionally active and pure parkin proteins. After evaluating the reliability of the method using DL-selenomethionine and DL-dithiothreitol as positive controls, this method was successfully applied to capture parkin ligands from Polygoni Cuspidati Rhizoma et Radix and Sophorae Flavescentis Radix. LC/MS identified seven novel parkin-targeting compounds, namely, 7,4′-dihydroxy-5-methoxy-8-(γ, γ-dimethylallyl)-flavanone, kushenol I, kurarinone, sophoraflavanone G, torachrysone-8-O-glucoside, apigenin, and emodin, supported by the molecular docking analysis. Five of the seven novel compounds (kushenol I, kurarinone, sophoraflavanone G, apigenin, and emodin) can activate parkin in in vitro autoubiquitination assays. Meanwhile, kushenol I and kurarinone had antisteatosis activity in fat emulsion-damaged human hepatocytes. These results confirmed the effectiveness of the method for identifying parkin ligands from complex preparations, useful to advance drug discovery from medicinal herbs.
Highlights
Parkin is an E3 ubiquitin ligase that localizes to the cytoplasm and mitochondria and plays a key role in the degradation of cytotoxic proteins through the ubiquitin-proteasome system
High-throughput screening methods [3] identified parkin ligands, these are often unsuitable for directly determining multiple ligands from complex mixtures
This study developed a rapid and efficient fishing method combining centrifugal ultrafiltration (CU) with Liquid chromatography/mass spectrometry (LC/MS) to identify parkin ligands from Polygoni Cuspidati Rhizoma et Radix (PCRR) and Sophorae Flavescentis Radix (SFR)
Summary
Parkin is an E3 ubiquitin ligase that localizes to the cytoplasm and mitochondria and plays a key role in the degradation of cytotoxic proteins through the ubiquitin-proteasome system. Parkin is a prominent pharmacological target for drug development. HMs, including Cinnamomum cassia Presl [1] and Rhodiola rosea L [2], regulate parkin expression and mitophagy. The classic procedure for discovering target compounds from HMs involves extraction and fishing, followed by the pharmacological screening of the purified substances. This method is time-consuming, labor-intensive, expensive, and often inefficient for directly screening bioactive compounds from natural samples. High-throughput screening methods [3] identified parkin ligands, these are often unsuitable for directly determining multiple ligands from complex mixtures. Further development of efficient strategies is required to identify specific parkin ligands from complex samples
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