Abstract
BackgroundIdentification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis.ResultsWe applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins.ConclusionsThis study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.
Highlights
Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by coomassie brilliant blue (CBB) staining and MS/MS analysis
Affinity chromatography is often used for identifying biological targets of multiple small molecules of interest; it is usually very difficult to identify compoundtargeted protein with low expression because of the low sensitivity of detection using coomassie brilliant blue (CBB) staining and MS/MS analysis
To overcome the limitations of affinity chromatography, we propose a new protocol combining in silico screening and experimental verification for identification of target proteins
Summary
Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. Several bioactive compounds have led to breakthroughs in understanding the functional roles of proteins [3,4,5,6,7,8,9,10,11]; one significant hurdle to developing new chemical probes of biological systems is. Affinity chromatography is often used for identifying biological targets of multiple small molecules of interest; it is usually very difficult to identify compoundtargeted protein with low expression because of the low sensitivity of detection using coomassie brilliant blue (CBB) staining and MS/MS analysis. To overcome the limitations of affinity chromatography, we propose a new protocol combining in silico screening and experimental verification for identification of target proteins
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