Identifying Drug Effects via Pathway Alterations using an Integer Linear Programming Optimization Formulation on Phosphoproteomic Data

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.Author Summary: Cells are complex functional units. Signal transduction refers to the underlying mechanism that regulates cell function, and it is usually depicted on signaling pathways maps. Each cell type has distinct signaling transduction mechanisms, and several diseases arise from alterations on the signaling pathways. Small-molecule inhibitors have emerged as novel pharmaceutical interventions that aim to block certain pathways in an effort to reverse the abnormal phenotype of the diseased cells. Despite that compounds have been well designed to hit certain molecules (i.e., targets), little is known on how they act on an “operative” signaling network. Here, we combine novel high throughput protein-signaling measurements and sophisticated computational techniques to evaluate drug effects on cells. Our approach comprises of two steps: build pathways that simulate cell function and identify drug-induced alterations of those pathways. We employed our approach to evaluate the effects of 4 drugs on a cancer hepatocytic cell type. We were able to confirm the main target of the drugs but also uncover unknown off-target effects. By understanding the drug effects in normal and diseased cells we can provide important information for the analysis of clinical outcomes in order to improve drug efficacy and safety.