Phenotypes assay

Cell microarrays have also been fabricated. Ziauddin and Sabatini (2001) demonstrated the ability to transfect cells cultured onto plasmid DNA arrayed in gelatin on a standard DNA microarray slide. Xu (2002) printed down cells in the form of high density microarrays on permeable membranes and demonstrated phenotypic assay performance with the immobilized cells. The commercialization of viable cell arrays will permit an even closer look at cell-mediated events during the drug discovery process. 

We do not address early toxicology profiling in this section, that is, those phenotypic assays for which molecular targets are hardly known and which attempt to be predictive of the standard later stage assays, such as those covering genotoxicity, hepatotoxicity or phototoxicity. These are all covered elsewhere in this book. 

TPMT activity can be analyzed by different phenotyping assays including the radiochemical method developed by Weinshilboum and more recent non-radioactive HPLC methods using either 6-MP or 6-TG as substrates ( 145, 146, 147, 148, 149,150,151,152,153). The radiochemical and HPLC assays have been shown to lead to comparable results with 6-MP as a substrate (146,147,148,149,150). However, when using 6-TG, TPMT activity was measured at 30% higher levels (152). 

Ford L, Graham V, Berg J. Whole-blood thiopurine 5-methyltransferase activity with genotype concordance a new, simplified phenotyping assay. Ann Clin Biochem 2006 43 354-360. 

Forward chemical genomics Phenotype or response-driven Traditional drug discovery Molecular target unknown or poorly characterized Phenotypic assays, organ bath, tissue binding assays, etc. Target for Probe ... 

Saraiva L, Fresco P, Pinto E, and Goncalves J (2004) Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay. European Journal of Pharmacology 491 101-110. 

Both genotypic and phenotypic drug resistance assays are valuable tools in this respect. Where genotypic tests are fast and relatively cheap to monitor the presence of known resistance-related mutations, they suffer from known and unknown synergistic and antagonizing effects of combinations of mutations. Only phenotypic assays can measure the actual inhibitory effects of the antiviral drugs on the clinical HIV-1 isolate. Specific reverse transcriptase (RT) and... 

In Subheading 5. a few genotypic tests will be described. Using phenotypic assays, clinical isolates that display various levels of resistance to particular drugs have been identified. Often the researcher wants to know what mutations are responsible for this phenotypic resistance, in an attempt to understand and possibly to anticipate the development of the resistance. Because present-day clinical anti-HIV drugs are RT or protease inhibitors, sequencing protocols for clinical HIV-1 RT and protease genes will be described. Once researchers have established a stable relationship between resistance and particular mutations, tests that identify specific mutations may be very valuable and much faster than any other method. In this respect, the protocol for two amplification refractory mutation systems (ARMS) will be described, which use mutation-specific PCRs. Other useful assays are the point mutation assay (10) and LiPA (see also Chapter 10). 

Kellam, P. and Larder, B. A. (1994) Recombinant virus assay a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates. Antimicrob. Agents Chemother. 38, 23-30. 

A limitation of the currently used genotypic and phenotypic assays is that they can detect only those mutants that make up at least 20% of the total viral population. Regimens chosen based on resistance testing may not always be effective because the minority populations will quickly predominate in the presence of a drug. Drug selection pressure is also needed for some resistance mutations to persist at detectable concentrations in the viral population when the drug therapy is discontinued, the wild-type virus may quickly predominate. For this reason, it is recommended that specimens for resistance testing be obtained while the patient is on antiretroviral therapy. 

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