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In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound.

Li L, Wijaya H, Samanta S, Lam Y, Yao SQ - Sci Rep (2015)

Bottom Line: Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery.Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action.We caution its potential therapeutic effects should be further investigated in detail.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, National University of Singapore, Singapore 117543 [2] Key Laboratory of Flexible Electronics (KLOFE) &Institute of Advanced Materials (IAM), National Jiangsu Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, P. R. China.

ABSTRACT
Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail.

No MeSH data available.


Targets validation.a, Silver-stained (SS) PD samples of in situAP1 (10 μM, 3 h)-labeled A549 cells. The cut band was subjected to LC-MS/MS analysis and results are summarized in b, Note: N, C, M and E represent nucleus, cytoplasm, mitochondrion and Endoplasmic reticulum, respectively. n/a = Not available. c, PD/WB target validation of A549 cells labeled by AP1 (10 μM, 3 h) with or without NEM treatment. d, Graphical summary of CETSA results of A549 cells treated with WT (100 μM, 3 h). WT/p50 interaction in HepG2 cells were used as a positive control. e, Cell imaging of A549 cells labeled by AP1 (10 μM, 3 h) followed by click chemistry with Rh-PEG-N3. IF = immunofluorescence (λex/em = 630/650–750 nm). (Insets) DIC images. Scale bar = 10 μm. f Concentration-dependent in vitro labeling of recombination proteins (~50 ng) by AP1 (1 h), with or without WT (100 μM). Full size WB can be found in Supplementary Fig. S13.
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f4: Targets validation.a, Silver-stained (SS) PD samples of in situAP1 (10 μM, 3 h)-labeled A549 cells. The cut band was subjected to LC-MS/MS analysis and results are summarized in b, Note: N, C, M and E represent nucleus, cytoplasm, mitochondrion and Endoplasmic reticulum, respectively. n/a = Not available. c, PD/WB target validation of A549 cells labeled by AP1 (10 μM, 3 h) with or without NEM treatment. d, Graphical summary of CETSA results of A549 cells treated with WT (100 μM, 3 h). WT/p50 interaction in HepG2 cells were used as a positive control. e, Cell imaging of A549 cells labeled by AP1 (10 μM, 3 h) followed by click chemistry with Rh-PEG-N3. IF = immunofluorescence (λex/em = 630/650–750 nm). (Insets) DIC images. Scale bar = 10 μm. f Concentration-dependent in vitro labeling of recombination proteins (~50 ng) by AP1 (1 h), with or without WT (100 μM). Full size WB can be found in Supplementary Fig. S13.

Mentions: With a long history of remedial applications in TCM, Andrographis paniculata is currently a World Health Organization (WHO)-listed herb33. Its active ingredient, WT, despite having relatively poor cellular activities (in potency and selectivity), is being pursued by many research labs as a promising drug candidate1819. We were intrigued by this natural product because most of its reported cellular and pharmacological properties point to the likelihood that it belongs to a group of molecules called pan-assay interference compounds (PAINS), which are highly promiscuous and appear active in many biological assays34. The in situ proteome profiling capability of AP1 made it possible to test this hypothesis (Fig. 4). We were also interested to positively identify the 60-kDa labeled band, which appeared to be the most prominent cellular target of WT in A549 cells (a human lung adenocarcinoma epithelial cell line). Large-scale PD/LC-MS/MS experiments were performed with AP1-labeled cells and the gel slice at ~60-kDa region was cut, tryptically digested and analyzed (Fig. 4a,b); a total of six high-confidence candidate proteins were identified, and three cytosolic proteins were further confirmed to be true cellular targets of WT (Fig. 4c–e). NAMPT is a pleiotropic enzyme involved in a number of human diseases35. ALDH1B1 and GSR are two key proteins required to balance endogenous reactive oxygen species (ROS)3637. PD/WB results indicate all three proteins were endogenously labeled in A549 cells even without NEM treatment. p50 on the other hand was positively labeled only in NEM-treated A549 cells. In HepG2 cells, they were labeled only after NEM treatment (Supplementary Fig. S10). In a cellular thermal shift assay (CETSA)38, all three proteins were efficiently stabilized by WT in A549 cells, indicating they were physically engaged in binding to WT in intake cells (Fig. 4d). p50 was similarly stabilized by WT in HepG2 cells. Cellular imaging of AP1-labeled A549 cells further indicates fluorescence signals from all three newly identified WT targets completely colocalized with those from the Rh-PEG-N3 channel (Fig. 4e, panels 3/6/9), but not vice versa, indicating there were additional endogenous AP1 targets. Finally, 6 recombinant proteins known to possess nucleophilic cysteine residues were randomly chosen and labeled by AP1 (Fig. 4f); results showed all but one were positively labeled at 1–10 μM (a concentration lower than most published WT protocols1819), but negated in the presence of excess WT. These results thus unequivocally confirm WT is indeed a highly promiscuous compound.


In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound.

Li L, Wijaya H, Samanta S, Lam Y, Yao SQ - Sci Rep (2015)

Targets validation.a, Silver-stained (SS) PD samples of in situAP1 (10 μM, 3 h)-labeled A549 cells. The cut band was subjected to LC-MS/MS analysis and results are summarized in b, Note: N, C, M and E represent nucleus, cytoplasm, mitochondrion and Endoplasmic reticulum, respectively. n/a = Not available. c, PD/WB target validation of A549 cells labeled by AP1 (10 μM, 3 h) with or without NEM treatment. d, Graphical summary of CETSA results of A549 cells treated with WT (100 μM, 3 h). WT/p50 interaction in HepG2 cells were used as a positive control. e, Cell imaging of A549 cells labeled by AP1 (10 μM, 3 h) followed by click chemistry with Rh-PEG-N3. IF = immunofluorescence (λex/em = 630/650–750 nm). (Insets) DIC images. Scale bar = 10 μm. f Concentration-dependent in vitro labeling of recombination proteins (~50 ng) by AP1 (1 h), with or without WT (100 μM). Full size WB can be found in Supplementary Fig. S13.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4478469&req=5

f4: Targets validation.a, Silver-stained (SS) PD samples of in situAP1 (10 μM, 3 h)-labeled A549 cells. The cut band was subjected to LC-MS/MS analysis and results are summarized in b, Note: N, C, M and E represent nucleus, cytoplasm, mitochondrion and Endoplasmic reticulum, respectively. n/a = Not available. c, PD/WB target validation of A549 cells labeled by AP1 (10 μM, 3 h) with or without NEM treatment. d, Graphical summary of CETSA results of A549 cells treated with WT (100 μM, 3 h). WT/p50 interaction in HepG2 cells were used as a positive control. e, Cell imaging of A549 cells labeled by AP1 (10 μM, 3 h) followed by click chemistry with Rh-PEG-N3. IF = immunofluorescence (λex/em = 630/650–750 nm). (Insets) DIC images. Scale bar = 10 μm. f Concentration-dependent in vitro labeling of recombination proteins (~50 ng) by AP1 (1 h), with or without WT (100 μM). Full size WB can be found in Supplementary Fig. S13.
Mentions: With a long history of remedial applications in TCM, Andrographis paniculata is currently a World Health Organization (WHO)-listed herb33. Its active ingredient, WT, despite having relatively poor cellular activities (in potency and selectivity), is being pursued by many research labs as a promising drug candidate1819. We were intrigued by this natural product because most of its reported cellular and pharmacological properties point to the likelihood that it belongs to a group of molecules called pan-assay interference compounds (PAINS), which are highly promiscuous and appear active in many biological assays34. The in situ proteome profiling capability of AP1 made it possible to test this hypothesis (Fig. 4). We were also interested to positively identify the 60-kDa labeled band, which appeared to be the most prominent cellular target of WT in A549 cells (a human lung adenocarcinoma epithelial cell line). Large-scale PD/LC-MS/MS experiments were performed with AP1-labeled cells and the gel slice at ~60-kDa region was cut, tryptically digested and analyzed (Fig. 4a,b); a total of six high-confidence candidate proteins were identified, and three cytosolic proteins were further confirmed to be true cellular targets of WT (Fig. 4c–e). NAMPT is a pleiotropic enzyme involved in a number of human diseases35. ALDH1B1 and GSR are two key proteins required to balance endogenous reactive oxygen species (ROS)3637. PD/WB results indicate all three proteins were endogenously labeled in A549 cells even without NEM treatment. p50 on the other hand was positively labeled only in NEM-treated A549 cells. In HepG2 cells, they were labeled only after NEM treatment (Supplementary Fig. S10). In a cellular thermal shift assay (CETSA)38, all three proteins were efficiently stabilized by WT in A549 cells, indicating they were physically engaged in binding to WT in intake cells (Fig. 4d). p50 was similarly stabilized by WT in HepG2 cells. Cellular imaging of AP1-labeled A549 cells further indicates fluorescence signals from all three newly identified WT targets completely colocalized with those from the Rh-PEG-N3 channel (Fig. 4e, panels 3/6/9), but not vice versa, indicating there were additional endogenous AP1 targets. Finally, 6 recombinant proteins known to possess nucleophilic cysteine residues were randomly chosen and labeled by AP1 (Fig. 4f); results showed all but one were positively labeled at 1–10 μM (a concentration lower than most published WT protocols1819), but negated in the presence of excess WT. These results thus unequivocally confirm WT is indeed a highly promiscuous compound.

Bottom Line: Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery.Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action.We caution its potential therapeutic effects should be further investigated in detail.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, National University of Singapore, Singapore 117543 [2] Key Laboratory of Flexible Electronics (KLOFE) &Institute of Advanced Materials (IAM), National Jiangsu Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, P. R. China.

ABSTRACT
Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail.

No MeSH data available.