Abstract
Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts). E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (Kd imm) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and-in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer-cellular assays. Taken together, the success of our strategy-which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry-in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.
| Original language | English |
|---|---|
| Pages (from-to) | 1495-1509 |
| Number of pages | 15 |
| Journal | Molecular and Cellular Proteomics |
| Volume | 13 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 2014 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Analytical Chemistry
- Medicine(all)
Cite this
Quantitative chemical proteomics identifies novel targets of the anti-cancer multi-kinase inhibitor E-3810. / Colzani, Mara; Noberini, Roberta; Romanenghi, Mauro; Colella, Gennaro; Pasi, Maurizio; Fancelli, Daniele; Varasi, Mario; Minucci, Saverio; Bonaldi, Tiziana.
In: Molecular and Cellular Proteomics, Vol. 13, No. 6, 2014, p. 1495-1509.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Quantitative chemical proteomics identifies novel targets of the anti-cancer multi-kinase inhibitor E-3810
AU - Colzani, Mara
AU - Noberini, Roberta
AU - Romanenghi, Mauro
AU - Colella, Gennaro
AU - Pasi, Maurizio
AU - Fancelli, Daniele
AU - Varasi, Mario
AU - Minucci, Saverio
AU - Bonaldi, Tiziana
PY - 2014
Y1 - 2014
N2 - Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts). E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (Kd imm) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and-in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer-cellular assays. Taken together, the success of our strategy-which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry-in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.
AB - Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts). E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (Kd imm) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and-in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer-cellular assays. Taken together, the success of our strategy-which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry-in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.
UR - http://www.scopus.com/inward/record.url?scp=84901950348&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84901950348&partnerID=8YFLogxK
U2 - 10.1074/mcp.M113.034173
DO - 10.1074/mcp.M113.034173
M3 - Article
C2 - 24696502
AN - SCOPUS:84901950348
VL - 13
SP - 1495
EP - 1509
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 6
ER -