Drug phenotypic assays

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. 

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 ... 

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. 

Petropoulos CJ, Parkin NT, Lhnofi KL, Lie YS, Wrin T, Huang W, et al. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 2000 44 920-8. 

He JH, Guo SY, Zhu F, Zhu JJ, ChenYX, Huang CJ, Gao JM, Dong QX, Xuan YX, Li CQ (2013) A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity. J Pharmacol Toxicol Methods 67, 25-32. 

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). 

The ultimate goal of lead optimization is to produce compounds that will elicit the desired cellular and organismal phenotype when dosed at appropriate concentrations. During the course of lead optimization activities it is common for pharmacologists to evaluate compounds not only using in vitro enzyme activity assays but also in cell-based assays as well. A question that often arises at this stage of drug discov-... 

Veenstra and colleagues have identified five criteria for evaluating the cost-effectiveness of pharmacogenomics severity of the outcome avoided, drug monitoring, genotype-phenotype association, assay, and polymor-... 

Phenotypic resistance assays directly measure the ability of HlV-1 to replicate in a cell culture in the presence of different antiretroviral drug concentrations. This process is similar to that used to determine antibiotic resistance and is, therefore, more familiar to most clinicians. The recombinant virus, composed of a virus s reverse transcripfase and protease genes, is inserted into a standard reference strain of virus. The recombinant virus is then tested in vitro for fhe amount of drug needed to inhibit virus replication by 50%, relative to the amount of drug needed to inhibit a reference strain of virus. Phenotypic resistance testing is limited by the fact that it is conducted in vitro and not in vivo. 

LC/MS/MS with selected reaction monitoring (SRM) offers a fast and simple means to analyze biological matrices, which is a key factor in high-throughput CYP inhibition screens using liver microsomes. Potentially, the LC/MS/MS technique is suitable for analyses of cocktail substrates in other in vitro drug metabolism evaluations such as CYP induction/activation assays, rapid analysis of pooled liver microsomes, rapid reaction phenotyping of tissue (hepatic and extrahepatic) samples, as well as evaluation of hepatocytes/tissue slice CYP activity. ° ... 

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