State-based discovery: a multidimensional screen for small-molecule modulators of EGF signaling

Abstract

As an alternative to conventional, target-oriented drug discovery, we report a strategy that identifies compounds on the basis of the state that they induce in a signaling network. Immortalized human cells are grown in microtiter plates and treated with compounds from a small-molecule library. The target network is then activated and lysates derived from each sample are arrayed onto glasssupported nitrocellulose pads. By probing these microarrays with antibodies that report on the abundance or phosphorylation state of selected proteins, a global picture of the target network is obtained. As proof of concept, we screened 84 kinase and phosphatase inhibitors for their ability to induce different states in the ErbB signaling network. We observed functional connections between proteins that match our understanding of ErbB signaling, indicating that state-based screens can be used to define the topology of signaling networks. Additionally, compounds sort according to the multidimensional phenotypes they induce, suggesting that state-based screens may inform efforts to identify the targets of biologically active small molecules.The signal transduction networks that control human cellular physiology typically comprise tens to hundreds of proteins that interrelate in a complex, nonlinear fashion. Defects in these systems underlie many human pathologies, including cancer 1 , autoimmunity 2 and developmental abnormalities 3 . One of the principal challenges of systems biology is to understand how information flows through these networks and how we can best intervene to halt or to redirect the flow of aberrant signaling. Small molecules that modulate the activity of signaling proteins are useful both as tools to dissect protein function and as potential therapeutics. Currently, most efforts to discover such compounds are target-based: active compounds are identified by their ability to modulate the function of a specific protein of interest. Altering the flow of information through a network, however, may require nonintuitive solutions; it may even require molecules that target more than one protein.To address this need, we developed a method that identifies compounds by their ability to induce different states in a network. Here we define the 'state' of a network as the quantitative levels of its components and assume that an informative picture can be obtained by measuring a subset of these components in cell lysates. We refer to this strategy as 'state-based discovery'. Having the ability to push networks into different states will provide us with tools to dissect how information is directed, and redirected, through these systems in real time.

Complexity of Receptor Tyrosine Kinase Signal Processing

“…"Reverse" cross talk, in which TrkA and TrkB receptors induce activation of Ret, was also observed (TsuiPierchala et al 2002;Esposito et al 2008). Although in this case positive regulation of Ret In a recent study (Gujral et al 2012), breast cancer samples were subjected to "reversephase" protein microarrays (Sevecka and MacBeath 2006) and phosphorylation or total protein levels of multiple signaling molecules were tested. The resulted data were analyzed by unsupervised hierarchical clustering (Herrero et al 2001), revealing 12 major clusters of correlating proteins.…”

Analytical strategies for phosphoproteomics

“…In a recent study by Sevecka and MacBeath, antibodies were combined with microarrays for the detection of the abundance and phosphorylation state of selected proteins in order to study ErbB signaling [26,27].…”

“…Tyrosine phosphorylation, in particular, is of very low abundance. In nature, the ratio of phosphoserine, phosphothreonine, and phosphotyrosine is reported to be 1800:200:1 [30], however, highly selective phosphotyrosine-specific antibodies exist and can be utilized for the enrichment of tyrosine phosphorylated proteins prior to analysis by MS [26]. Pandey et al used this approach to characterize the epidermal growth factor (EGF) receptor signaling pathway and they identified a novel immunoreceptor tyrosine-based activation motif-containing protein (STAM2) and characterized its role in growth factor and cytokine receptor signaling pathways.…”

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling

“…Tools for analyzing interconnectivity of biological molecules have so far been used primarily to explore system-level gene expression data. However, such computational methods may also elucidate how kinase activity information is directed, and by using these algorithms, we assumed that phosphorylation events that appeared simultaneously might be interlinked and provide information about pathway connectivity [18,19].…”