A Quantitative Chemical Proteomics Approach to Profile the Specific Cellular Targets of Andrographolide, a Promising Anticancer Agent That Suppresses Tumor Metastasis

Abstract

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug's current therapeutic regime and expand the drug's therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drugbinding sites of two protein targets, NF-B and actin. Molecular & Cellular Proteomics 13: 10.1074/mcp. M113.029793, 876-886, 2014.As most drugs exert pharmacological effects by interacting with their target proteins, the identification of these target proteins is a critical step in unraveling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting potentially unrevealed actions and thus refining future clinical applications of the substance. Traditional approaches used to identify protein targets of a drug typically utilize immobilized drug affinity chromatography coupled with mass spectrometry (MS) 1 (1, 2). These methods can be applied to cell lysates, but not in an in vivo setting, because of the requirement of a solid support. In vitro target profiling might not accurately reflect the drug's actions in the in vivo physiological environment. To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3-15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups. With the increasing sensitivity of modern MS platforms, low-abundance protein targets can be successfully identified. Although both conventional affinity chromatography and recent ABPP-based methods allow us to detect a set of candidate protein targets for a drug, it remains difficult to 1 The abbreviations used are: MS, mass spectrometry; ABPP, activity-based protein profiling; ICABPP, clickable activity-based protein profiling; iTRAQ, isobaric tags for relative and absolute quantitation; DMSO, dimethyl sulfoxide; Andro, androgr...

Andrographolide Restores Steroid Sensitivity To Block Lipopolysaccharide/IFN-γ–Induced IL-27 and Airway Hyperresponsiveness in Mice

“…Andrographolide is best known for its anti-inflammatory activity as an NF-kB inhibitor. It binds specifically to the DNA binding subunit p50 at Cys 62 residue of the NF-kB complex, preventing the complex from binding to DNA for gene transcription (39). It has been shown that andrographolide suppressed LPS-induced p65 phosphorylation, p65 nuclear translocation, and NF-kB DNA binding activity in mouse MEL-12 epithelial cells and Raw 264.7 cells (40,41).…”

Nanoemulsion as a strategy for improving the oral bioavailability and anti-inflammatory activity of andrographolide

“…9 Andrographolide (AG; Figure 1) is a natural diterpenoid isolated from Andrographis paniculata, which is a traditional remedy for fever, cold, sore throat, and various chronic infectious diseases. [10][11][12] Moreover, previous studies have shown that AG possesses many pharmacological activities, including anti-inflammatory, 13 antioxidative, 14 and anticancer 15 activities. A previous study showed that pretreatment with the ethanolic extract of A. paniculata provided significant protection against gastric ulcers.…”

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

“…An iTRAQ quantitative proteomics approach was used in tandem with BONCAT to differentiate any potential background contamination from selective enrichment, 29,30 to enhance the reliability of the results obtained. The background contamination includes the pre-existing proteins nonspecifically binding to the beads, as well as the endogenously biotinylated proteins, since streptavidin beads were used for the protein enrichment.…”

“…The iTRAQ labeling technique 29,30,48 was employed to investigate the changes in the proteomic profiles upon autophagy induction, per the manufacturer's instructions with minor modifications. Briefly, the dried digested peptides were resuspended in equal amounts of dissolution buffer (0.5 M TEAB; Sigma-Aldrich, T7408).…”

“…We have previously utilized iTRAQ to differentiate the specific binding proteins from the nonspecific ones during affinity enrichment. 29,30 In the current study, via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The functions of these proteins were deduced based on subsequent bioinformatics and pathway analysis and the trends of alterations in them (promoting or inhibiting) were predicted.…”