Tracing pathway activities with kinase inhibitors and reverse phase protein arrays

Abstract

Controlling aberrant protein kinase activity is a promising strategy for a variety of diseases, particularly cancer. Hence, the development of kinase inhibitors is currently a focal point for pharmaceutical research. In this study we utilize a chip-based reverse phase protein array (RPA) platform for profiling of kinase inhibitors in cell-based assays. In combination with the planar wave-guide technology the assay system has an absolute LOD down to the low zeptomole range. A431 cell lysates were analyzed for the activation state of key effectors in the epidermal growth factor (EGF) and insulin signaling pathways to validate this model for compound screening. A microtiter-plate format for growing, treating, and lysing cells was shown to be suitable for this approach, establishing the value of the technology as a screening tool for characterization of large numbers of kinase inhibitors against a wide variety of cellular signaling pathways. Moreover, the reverse array format allows rapid development of site-specific phosphorylation assays, since in contrast to ELISA type systems only a single antigen-specific antibody is required.

Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

“…RPPA technology is particularly well suited to drug and biomarker discovery as a result of the high-sensitivity, high-reproducibility, high-throughput, and quantitative features of the approach. Recent studies have applied RPPA to determine the mechanism of action and selectivity of emerging drug candidates at the pathway level (82), as well as to uncover unexpected drug resistance mechanisms (83). Dose-and time-dependent profiling of pathway responses via RPPA following exposure of cultured cells to candidate drugs enables precise calculation of the potency upon key signaling molecules.…”

“…Correlation of potency (e.g. EC 50 ) values across multiple analytes at sequential time points following drug addition enables discrimination of off-target activities from downstream signaling and pathway cross-talk events (82,84).…”

Targeting PI3K/mTOR Overcomes Resistance to HER2-Targeted Therapy Independent of Feedback Activation of AKT

“…Tumors were excised, divided in half and either snap-frozen in liquid nitrogen, or prepared as formalin-fixed/paraffin-embedded (FFPE) tissues. Tumor and serum drug concentration determination and reverse phase protein arrays (RPPA) were performed as previously described (28,34). All animal work was carried out under a protocol approved by the Institutional Animal Care and Use Committee and the University of California, Los Angeles Animal Research Committee.…”

Multiparametric Analysis of Screening Data: Growing Beyond the Single Dimension to Infinity and Beyond

“…Recently, application of topological data analysis to multiparametric data led to the development of t-SNE (t-distributed stochastic neighbor embedding), 16,66 a method in which multiparametric data are projected in a 2D space in a way that preserves the relations among the data points. An implementation of this technique, called viSNE, has recently been used to analyze results of singlecell analysis, highlighting similarities among healthy donors and their differences from patient-derived samples.…”

“…Methods that focus on monitoring the presence of a number of preselected proteins using affinity reagents such as antibodies or aptamers have a higher throughput and so can be used for screening applications. Such methods include technologies such as protein arrays 16,17 or Luminex bead-based multiplexed assays. 18 Recent developments have enabled the use of fluorescenceactivated cell sorting (FACS) for multiparametric screening.…”