Utilizing a novel, chemical proteomics approach, we have identified 577 proteins which interact with the biotinylated SO, TP-304, in cell culture. While some of these proteins had previously been identified, to date, this is the first report of a comprehensive and system-wide analysis of SO binding partners. As expected, a large number of new binding partners were identified, forming a large interconnected network of proteins that interact with SO. Many of the canonical pathways previously implicated as being targeted by SO, such as NFκB, JAK/STAT, PTEN and AMPK were found in this analysis 
In addition to these networks identified in silico, we have functionally validated the mTOR pathway as a target for CDDO-Im. Previous studies have suggested that this pathway may be inhibited by very high concentrations of the SO, CDDO-Me 
. However, this is the first report to show that CDDO-Im also inhibits this pathway and also the first report that nanomolar concentrations of an SO directly inhibit the kinase activity of mTOR. mTOR belongs to a family of 6 kinases referred to as phosphoinositide-3-OH-kinase-related kinases (PIKKs). Interestingly the PIKK family members ATM and ATR and TRRAP were also pulled-down by TP-304.
The SO are extremely potent inhibitors of cellular proliferation 
. The concentrations at which such inhibition is observed correlates with those we report for mTOR inhibition. Therefore, one may propose that this potent inhibition of proliferation may, in part, be due to inhibition of translation through disrupting mTOR activity. The identification of the PI3K/mTOR pathway as a central node in the STRING analysis interconnecting critical cell cycle and cell division proteins as well as the prominence of cell cycle proteins in the GO analysis would suggest this may be an important target for the SO. Also, it was recently reported that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170 which is an mTOR target protein 
. Our observations suggest that the mechanism by which CDDO-Im disrupts Clip-170 may be through inhibition of the kinase activity of mTOR. Aberrant activation of mTOR and S6K has been shown to play a critical role in the development of diabetes and diabetic nephropathy 
. Currently, CDDO-Me (Bardoxolone methyl) is undergoing late stage clinical development for the treatment of nephropathy in diabetes patients and significant improvements in markers of renal function have been reported 
. Some of the improvements observed in these patients may be mediated through the inhibition of the mTOR/S6K signaling axis. Further studies toward this goal are ongoing in our laboratory.
One caveat of our proteomics-based target search is its qualitative nature, which lacks quantitative information about the SO-target interaction. Binding in such assays is a function of both protein/target abundance and affinity; therefore, highly abundant-low affinity proteins will co-purify with low abundance high-affinity proteins, making it difficult to infer relative affinities of the SO for the identified targets. However, multi-functional drugs typically bind to targets with lower affinity than a single target drug. Previous studies have suggested that low-affinity, multi-target drugs have a lower prevalence and a reduced range of side-effects than high-affinity, single-target drugs while also being more efficacious 
. By virtue of their low affinity binding, drugs of this type work best under pathological conditions, for the prevention of disease by restoring cellular homeostasis. Indeed it is in the area of chemoprevention that the SO have excelled in in vivo
models. Currently the development of network-based, multi-target drugs similar in function to the SO is gaining increased interest in the field of drug development 
. Also, it should be noted that not all 577 proteins identified by MS analysis may be direct SO targets. It is possible that the lysis and wash conditions used did not completely disrupt high affinity protein complexes. We identified numerous proteins that are known to form high affinity complexes in cells including, but not limited to ATM/ATR and mTORC1. While this may cloud the identification of direct targets it facilitates the identification of protein complexes and pathways with which the SO interacts. One other caveat is the possibility that the coupling of the biotin group on the btSO may impede interactions with some target proteins. Indeed, TP-304 appears to have a lower affinity for KEAP1 (unpublished observation) which explains its absence from our list of proteins identified by MS. However, in spite of the above caveats, the data shown here open the way to new investigations that will examine the more difficult problem of interactions of SO with targets in real time.
In summary, technological advances in the integration of proteomics, pharmacology and molecular biology now provide the means to map drugs and targets at a systems level. Indeed, it has become evident that many drugs thought to specifically target a single protein, e.g.
the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib, are, in fact more promiscuous than originally thought 
. Further advances in drug development, enhanced by an understanding of the genetic and epigenetic complexity of many chronic diseases, will come from drugs that target multiple components of critical signaling pathways and metabolic networks. In this regard, our studies demonstrate the utility of proteomic methods using biotinylated compounds as probes for identifying new candidate targets and perhaps may facilitate the use of SO in previously unrecognized therapeutic applications. The goals of targeting complex networks and applying systems biology to drug discovery are now of immediate importance.