Abstract
Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.
Highlights
Identifying the protein targets of therapeutic natural products and deciphering the specific mechanisms of action at the molecular level are crucial steps in the development of natural products as drugs to treat human diseases [1,2]
We investigated the mode of action of voacangine via label-free drug affinity responsive target stability (DARTS) and successfully identified vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein responsible for the observed antiangiogenic properties of voacangine in endothelial cells (ECs)
We performed a DARTS assay to validate the interaction between curcumin and aminopeptidase N (APN) (Figure 1a)
Summary
Identifying the protein targets of therapeutic natural products and deciphering the specific mechanisms of action at the molecular level are crucial steps in the development of natural products as drugs to treat human diseases [1,2]. Widely-applied approaches include affinity-based matrices that label or tag small molecules, such as affinity pull-downs and phage display methods [5,6,7] These methods have limitations, such as changes in structural properties may occur upon labeling with chemical probes, or upon tagging functional groups for immobilization; biological activities of natural products may change as a result of the alterations in the chemical structure; these processes incur a high cost, and are time and labor-consuming; and difficulty in modifying small molecules due to availability of only virtual three-dimensional structures [8,9]. Through unbiased DARTS approaches in combination with mass spectrometry (MS) analysis, quantitative MS based-proteomics is utilized to identify multiple target proteins in drug-treated versus control samples [15]
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