The clinical success of imatinib in chronic myelogenous leukemia underscored the utility of kinase inhibitors in treating cancers (Druker et al., 2001
). A variety of small molecules are now being screened to identify cancer therapeutic agents that are directed against kinases and other key signaling molecules. Although anti-cancer effects of some of the inhibitors are established, the proteins or pathways targeted by these inhibitors remain ill-characterized. Even when the direct targets of these inhibitors are established, the cellular networks that are affected by these inhibitors remain unexplored. It becomes important to characterize these as the kinases themselves are known to mutate thus becoming resistant to the drugs. Quantitative phosphoproteomics is especially useful in characterizing targets of kinase inhibitors. It facilitates exploring the effect of kinase inhibitors on entire signal transduction network at the resolution of individual phosphorylation sites. Bose et al.
utilized three state SILAC strategy to explore the effect of EGFR and Her2 selective inhibitor, PD168393 on Her2 overexpressing cells (Bose et al., 2006
). Similar study has also been undertaken to characterize the effect of two widely used MAPK inhibitors, U0126 and SB202190 on cellular phosphoproteome (Pan et al., 2009
). The same study also demonstrated nearly thousand phosphorylation events that were affected by dasatinib, a clinical drug against Gleevec-resistant, point mutated versions of BCR-Abl. While the effect of selective kinase inhibitors on cellular phosphoproteome is being studied using the above-mentioned strategies, efforts are also on to characterize the total kinome complement of cells and their phosphorylation dynamics.
Small molecule kinase inhibitors often target conserved ATP binding sites on kinases. Because of high conservation of this site across kinases, most inhibitors are not so specific and target multiple kinases. The selectivity of such inhibitors is usually assessed by in vitro
enzymatic assays using a set of recombinant protein kinases (Bain et al., 2003
; Davies et al., 2000
). However, these kinases are often expressed in non-human systems and may not truly reflect the behaviour of native kinases both in terms of folding as well as activity. Further, inhibitor selectivity is often tested across only a subset of kinases leaving the large majority untested. This has prompted researchers to look for alternative approaches by which target selectivity can be established for kinase inhibitors. An approach that will reveal the true (and broad) specificity of kinase inhibitors in physiological context is preferred. Recent advances in chemical proteomics involving kinase inhibitor based affinity matrices have opened up avenues for better characterization of cellular targets (Bantscheff et al., 2007
; Godl et al., 2003
By using immobilized SB 203580 analogue, a widely used p38 inhibitor, as bait, Godl et al. identified previously unknown targets in HeLa protein lysates (Godl et al., 2003
). Rip-like interacting caspase-like apoptosis-regulatory protein was more potently inhibited by SB 203580 as compared to p38. By employing immobilized nonselective kinase inhibitors (kinobeads) coupled with iTRAQ based quantitative proteomic strategy, Bantscheff et al. profiled protein targets of widely used ABL kinase inhibitors imatinib, dasatinib and bosutinib in K562 cells. In addition to confirming known targets including ABL and SRC family kinases, this strategy identified receptor tyrosine kinase DDR1 and oxidoreductase NQO2, as additional targets of imatinib (Bantscheff et al., 2007
). Mass spectrometric analysis of kinobead purifications from 14 human cell lines and tissues led to enrichment of 269 protein kinases, about 50% of all the kinases known in human. Interestingly, Rix et al. carried out a similar study employing three potent BCR-ABL inhibitors, imatinib, dasatinib and nilotinib and probed for cellular targets in K562 and primary chronic myeloid leukemia cells. These studies also identified DDR1 and oxidoreductase NQO2 as potent targets of these kinase inhibitors (Rix et al., 2007
). While off-targets in the kinase family are understandable, the identification of non-kinase target NQO2 by both the groups reveals unsuspected mechanisms through which some of the kinase inhibitors could be acting. These studies have established chemical proteomics using kinase enrichment matrices as useful strategies for identifying cellular targets of kinase inhibitors. This strategy is now increasingly employed to characterize several novel kinase inhibitors (Remsing Rix et al., 2009
; Rix et al., 2010
). Coupling kinase enrichment strategy with phosphoproteomics will not only identify cellular targets of kinase inhibitors, but will also reveal endogenous phosphorylation status of the enriched kinases that may reflect its activation status (Daub et al., 2008
; Oppermann et al., 2009
) (). In another approach, Hahn et al. integrated immunoaffinity-based tyrosine phosphoproteome profiling by mass spectrometry and RNAi based signature screening to identify candidate gefitinib targets for AML therapy (Hahn et al., 2009
). This approach identified SYK as a candidate target for gefitinib, a widely used EGFR inhibitor.
Identification of cellular targets of kinase inhibitors