Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1α) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on pathways, the cornerstone for identifying mechanisms of drug's efficacy.

The analysis presented herein, leveraged high throughput proteomic data via an ILP formulation to gain new insight into chondrocytes signaling and the pathophysiology of degenerative diseases in articular cartilage.

Construction of signaling pathway maps and identification of drug effects are major challenge for pharmaceutical industries. Signaling maps are usually obtained from manual literature search, automated text mining algorithms, or canonical pathway databases (i.e. Reactome, KEGG, STKE, Pathway Studio, Ingenuity etc.) and in some cases they are used in combination with gene expression or mass spec data in an effort to create pathways specific to cell types or diseases. Our approach combines computational models with novel multicombinatorial high-throughput phosphoproteomic data for the functional analysis of signalling networks in mammalian cells. On the experimental front, we subject the cells with hundreds of co-treatment with a diverse set of ligands and inhibitors and we measure phosphorylation events on key signaling proteins using the xMAP technology. On the computational front, we create pathway maps that are cell type specific by fitting our phosphoprotein dataset into generic signaling maps via an Integer Linear programming formulation. To identify drug effects, we monitor the differences of topologies created with and without the presence of drug. In the present work, we use this approach to identify the effects of Nilotinib, a well known anti-cancer drug.

Chondrocytes, the only cell type in articular cartilage, are responsible for maintaining the composition of cartilage extracellular matrix (ECM) through a complex interplay of anabolic and catabolic stimuli. Although understanding the way chondrocytes respond to stimuli is of utmost importance for shedding light into the etiology of joint diseases, an integrative approach to studying their signaling transduction mechanisms is yet to be introduced. Herein, we propose an approach that combines high throughput proteomic measurements and state of the art optimization algorithms to construct a predictive model of chondrocyte signaling network, downstream of 78 receptors of interest.