Introduction

Drug discovery program progression from "hit" compounds derived from high-throughput screening to identification of a lead drug candidate suitable for an Investigational New Drug, enabling toxicology studies (hit to lead), requires medicinal chemistry compound optimization driven by relevant pharmacological data. Pharmacological data regarding the mechanism of compound interaction with the molecular target, such as receptor agonists and antagonists, enzyme inhibition kinetics, and protein-ligand binding are clearly important. Quantitative methods for evaluating the effect of pharmacological agents on receptor activation and inhibition, enzyme kinetics, and ligand-receptor binding are reviewed in other chapters. Pharmacological targets adapted to high-throughput methods are often poor predictors of efficacy in vivo but are used for ease of testing (Fig. 3.1).Cell-free systems consisting of purified target protein, test compounds, and the necessary substrates and ligands for target protein function and signal generation are often the choice for primary, high-throughput compound screening. Cellfree screening eliminates cell metabolism, cell permeability, and protein binding issues. In the next least complicated system, cells for study are often transfected with the target gene or a reporter system of gene function in a manner that creates a wide assay window but also alters the ratio of the target receptor to cellular components. Such complex interactions, such as those in multiple-protein signaling pathways, are difficult to reconstruct in either cell-free systems or those employing engineered cells. On the other hand, animal and human tissues and whole organism studies cannot be carried out with sufficient throughput to successfully drive hit to lead optimization. Finally, untransfected mammalian cells provide intact signaling pathways and other complex mechanisms not reliably reproduced in cell-free systems or in cells with overex-pressed targets. Species-specific compound activities have been observed in drug discovery caused in part by differences in the amino acid

Humans

Humans

Ease cf assay

Figure 3.1. The ease compound testing is inversely proportional to the complexity of the system and the physiological relevance of the assay.

Ease cf assay

Figure 3.1. The ease compound testing is inversely proportional to the complexity of the system and the physiological relevance of the assay.

sequence of the target protein. An infamous example is provided by the discovery of a mouse G-CSF signal transduction pathway activator (1).This compound was identified in a high-throughput assay in murine cell line. Despite active research, medicinal chemistry efforts have not resulted in a compound active towards human G-CSFR. Thus, untransfected human cells provide the most relevant practical drug-screening systems.

The role of high-throughput pharmacology in the hit to lead compound candidate identification process includes determination of efficacy and evaluation of potential compound liabilities (solubility, adsorption, toxicity) and specificity of action. Other examples include data obtained from pharmacological profiling of lead compounds against a broad panel of natural human cells. It is most useful if these cells have been previously characterized for their response to known pharmacological agents. In vitro assays for evaluation of the adsorption and metabolism properties of compounds have been developed. Systems and methods that provide high definition, content-rich information about cellular responses and are robust enough to have proven useful in secondary testing pharmacological properties of compounds are reviewed in this chapter.

Rapid, High Content Pharmacology

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