Many drugs are inhibitors of enzymes involved in mediating the disease processes. Understanding the mechanism of action (MOA) of the target enzyme is critical in early discovery and development of drug candidates through extensive Structure-Activity Relationship (SAR) studies. This chapter contains a primer on the MOA of enzymes and its significance in drug discovery, types of inhibition, development and validation of MOA assays, data analysis and guidelines for performing these assays. New and experienced investigators will find this chapter useful when starting new projects involving enzyme targets.

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems-level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS-based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine-directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems‐level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS‐based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine‐directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays.

Advances in protein phosphorylation analysis by mass spectrometry (MS) are enabling the generation of high quality, quantitative datasets of protein phosphorylation with a breadth of coverage and reproducibility not previously attainable. Comparisons of signaling responses in cells at a network level are now feasible and studies looking at cellular response to ligand stimulation, drug treatment or genetic modification are transforming our understanding of how cellular decision processes are encoded through the signaling network. The large and dynamic datasets acquired through MS-based phosphoproteomics can be combined with other types of biological data for computational modeling of cellular decision processes with direct biological relevance to cellular state and predictive of cellular response. Signaling analysis at a network level is just beginning. Challenges remain in validating and translating initial models generated using defined in vitro models to in vivo systems. The advent of higher throughput methods for validating models generated with MS will deepen our understanding of the relationship between signaling and disease and therefore the development and implementation of therapeutics.

3508 Tyrosine phosphorylation, a critical regulator of cellular signaling cascades, is dysregulated in various diseases, including cancer. Phosphorylation pathways overlap, intersect and feedback, creating complex signaling networks that requires a broad systems-level analysis to determine relevant phosphorylation patterns or “signatures” for defining disease models. An antibody array platform, with a peptide-based immunoassay design, was developed to detect 45 tyrosine phosphorylation sites on 34 different proteins simultaneously, in a single sample (Ti-Tyr™ Chip, Epitome Biosystems, Inc). As aberrant EGFR signaling has been implicated in various types of human cancers, we used this antibody array to identify and quantify time-dependent changes in phosphorylation downstream of the EGF receptor in HeLa cells, stimulated with EGF. Changes in site-specific phosphorylation of downstream targets were quantified for important regulators of migration (PLC gamma, FAK), cell proliferation (Her2, Shc, Crk, Erk) and receptor endocytosis /degradation (Cbl, Ack). Temporal and quantitative analysis demonstrated differences in EGF-induced p38 and Erk phosphorylation profiles. In addition, the cell surface receptor tyrosine kinases, EGFR and Her2, showed different temporal activation profiles suggestive of EGFR transactivation of Her2. Peptide-based immunoassays for tyrosine phosphorylation can be used for temporal and quantitative analysis of phosphorylation for profiling receptor tyrosine kinase signaling, at a site-specific level. Changes in tyrosine phosphorylation “signatures” can be used to monitor responses to pathway stimulation and therapeutic interventions.

Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor-stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications.

Broad screening of protein phosphorylation has wide‐ranging utility for understanding the impact of disease or drug treatment on cell signaling pathways. To address this need, we have developed an antibody array for measuring tyrosine signaling across 45 phosphotyrosine sites across multiple signaling pathways (Ti‐Tyr ™ Profiling Chip). The approach utilizes a novel strategy of measuring peptide fragments liberated by protease digestion of the sample prior to analysis, and allows for both multi‐site profiling and absolute quantification using synthetic peptide standards. The utility of the Ti‐Tyr™ chip is demonstrated by measuring specific modulation of pathway phosphorylation in stimulated cells in the presence and absence of distinct kinase inhibitors. Phosphorylation changes detected in chip targets correlate with known biological effects using comparable conditions. The use of peptide standards on the chip allows for an absolute quantitative assessment of the same stimulation conditions or inhibitor treatments across different cell lines, for example EGF stimulation in A431 and PC‐3 cancer lines. The Ti‐Tyr ™ Tyrosine Chip should improve the ability to understand the effects of cell stimuli, the underlying basis of disease and the impact of drug treatments across multiple cell pathways and cell types.

This chapter discusses development of antimalarial inhibitors of hemoglobinases. Malaria is the fourth largest infectious disease in the world, behind diarrhoeal diseases, tuberculosis, and measles. Caused by protozoan parasites of the genus Plasmodium, the disease is transmitted to the human host by the bite of the female Anopheles mosquito. There are estimated to be between 300 to 500 million clinical cases of malaria per year, with an estimated mortality of 1.5 to 2.7 million per year. The majority of these deaths are in children under the age of five. There are several factors which have caused increased concern regarding the transmission and spread of malaria. One factor is the rapid and unprecedented movement of naive human populations into endemic areas. Global warming has the potential for both disrupting current patterns of transmission of malaria and in potentially expanding the range of the Anopheles mosquito. The driving force for concern, however, is the increasing resistance to current antimalarial chemotherapy. The chapter focuses on recent advances in the development of protease inhibitors as potential therapeutics for the treatment of malaria. Biology of hemoglobin degradation is elaborated and combinatorial library synthesis for inhibitors of plasmepsins is also explained.