Typical Phenotypic Assays

Typical Phenotypic Assays 10.43.1 Cell-Viability Assay... 

Other assays typically employed in the course of a drug design project include physicochemical binding assays (based on, for example, NMR or Biacore surface plasmon resonance methodologies), mode-of-action cell assays (e.g., employing a specific antibody-based biomarker readout of the target or a downstream target), and phenotypic assays (e.g., cell proliferation assays). 

Based on the results from those cell calibration assays, the routine incubation period was set at 3 days. During that interval, some of the cells will proliferate, some will be quiescent but metabolically active, and some will die, which is the case for most primary cell cultures. Because these cells are cultured for periods far shorter than their typical population doubling time (about 6 to 7 days), these cells should not fully adapt to the tissue culture conditions and, therefore, maintain their primary cell phenotype. 

Smaller compound sets and focused compound libraries can be used to screen more broadly for physiological phenotypes. In the typical high-content screening experiment, the effects of compounds on cellular assays are captured... 

The diagnosis of homocystinuria is based on the recognition of the clinical phenotype in conjunction with the identification of an elevated total plasma homocysteine and elevated plasma methionine concentrations (via quantitative plasma amino acid analysis). Low cystine and low cystathionine are also seen (Box 14.3). In addition, increased urinary excretion of homocysteine as well as cysteine-homocysteine disulfide can be identified on urine amino acid analysis. Confirmation of the diagnosis can be done via enzyme assay, typically performed on cultured skin fibroblasts, lymphocytes, or liver tissue, or via molecular studies. 

A cell assay is defined as measurement and analysis of cellular response to chemical and/or physical stimulus. Cellular responses are diverse alterations of intracellular and extracellular biochemistry, cell morphology, motility, and growth properties. These responses characterize the cell phenotype and are typically monitored in a culture dish or a multiwell plate, while more recently microfluidic devices have been employed. A cell assay performed in a microfiuidic device is sometimes termed an on-chip assay. 

It provides a basis to design a robust assay and how to interpret the outcome of the resulting pharmacologically relevant screen. Typically, the phenotypes caused by small molecules result in a cascade of responses. The clearly defined phenotype is necessary for hit selection and follow-up studies. 

In addition to enzyme assays, HDAC inhibitors are usually evaluated in cancer cell lines, where they potently inhibit growth proliferation to provide a cell-based phenotypic readout of activity. The cellular potency of HDAC inhibitors can exceed their activity in enzyme assays, as the precise HDAC isoforms that drive proliferation in a given cell type may be unknown. In any case, growth inhibition data should be supplemented with evidence to confirm that it is due to HDAC inhibition and not off-target effects. Typically, confirmatory evidence involves Western blotting of client proteins such as histones for nuclear HDACs or tubulin for HDAC6 to detect an increase in acetylation levels, or changes in a downstream biomarker such as induction of the cyclin-dependent kinase inhibitor p21. 

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