Inhibition assay

Both xenobiotics and endogenous substrates are primarily metabolized by a super-family of enzymes, named cytochrome F450s (CYP450s, 

Recently, Chu et al. reported an ultra-fast LC/MS method for analysis of cytochrome P450 3A4 and 2D6 inhibition assaysd Testosterone and dextromethorphan were used as the specific substrates for CYP3A4 and CYP 2D6, respectively. LC/MS analyses were performed on a Sciex API 3000 mass spectrometer equipped with a Shimadzu LC-lOAdvp pump and a PE 200 autosampler. A Phenomenex Luna CIS (4.6x30 mm) column was used along with very steep gradients. Each sample analysis was completed in 0.5 min. 

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. °  

Compound stability in a variety of matrices including varying pH buffers, plasma, and liver microsomes is an important consideration related to the success of the compound being developed in the clinic. Low stability profiles in these environments will lead to poor pharmacokinetics of a potential candidate. These stability results can be used by medicinal chemists to optimize the labile portion of the structures to further improve stability. 

Among the new techniques that have been developed to improve the throughput, turbulent-flow chromatography (TEC) coupled with MS has 

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After screening for toxicity, identification and/or quantification assays may need to be carried out if the screening method is not specific for the cyanobacterial toxin(s) under investigation. Suitable assays for these purposes include the physicochemical assays, HPLC, MS, and CE, and to some extent the immunoassays and protein phosphatase inhibition assays summarized in Section 2. 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

Inhibition values quoted in this chapter are those from enzyme inhibition assays. 

Oseltamivir carboxylate 18 is used in sialidase inhibition assays Numbering used is that of A/N2 Abed et al. (2008)... 

Several results are quite apparent from the data shown in Table II. It is evident from the pentapeptide model compounds that substitution of amino acid residues at positions 4 and 5 does not significantly affect the structure about the N-terminus. This observation corroborated earlier work from agglutination-inhibition assays, which demonstrated that the nature of the amino acid at position 4 of the peptide (or glycopeptide) is not a requirement for specificity. 

TABLE 5.1 IC50 (nM) Values from Cell-Growth Inhibition Assays... 

FIGURE 5.35 Growth inhibition assay of three human cancerous cell lines, with dendritic prodrug 38 in the presence ( ) and absence ( ) of PGA cells were incubated for 72 h. 

Nitroheteroarenes continue to attract research activity. A series of 5-nitro-2-furyl derivatives were evaluated for in vivo efficacy against T cruzi [64] and an improved toxicity profile. Compound 65 showed a good efficacy profile in vivo and better acute toxicity profile when compared to nifurti-mox. A series of 5-nitroindazoles, represented by 66 (IC50 = 7.4 iM), were reported as having activity similar to nifurtimox (IC50 = 3.4 iM) in a growth inhibition assay [65]. 

Direct detection is usually preferred in applications, where direct binding of analyte of concentrations of interest produces a sufficient response. If necessary, the lowest detection limits of the direct biosensors can be improved by using a sandwich assay. Smaller analytes (molecular weight < 10,000) are usually measured using inhibition assay, Figure 14. 

Figure 14. Detection formats used in affinity biosensors based on spectroscopy of guided waves a) direct detection, b) sandwich assay, and c) competitive inhibition assay.

Figure 11. Comparison of different assay types using a direct detection scheme were the receptors immobilized to the surface and the analyte is recognized at the surface (direct optical detection and using labelled systems), a competitive test scheme were labelled analyte molecules compete with the non-labelled sample, and thirdly a binding inhibition assay were analyte derivatives (ligand derivatives) are immobilized at the surface, in a preincubation phase the ligands block receptor molecules, non-blocked receptors go to the surface being either labelled or optically detected.

A typical example of such parallelized measurements is given in Figure 20. For a variety of inhibitors, the binding inhibition assay is given for a thrombin inhibition Figure 20 shows the binding curves obtained in parallel from 96 wells37. 

Figure 21. By combinatorial chemistry, 21 15-mer-peptides are synthesized, each shifted by one position of the aminoacid. In binding inhibition assay, an epitope mapping of the antibody is achieved.

Despite a considerable decrease in its inhibitory activity in the solid-phase inhibition assays (with an IC50 of 300 nM, corresponding to a 50-fold increase compared with 160), an equivalent decrease in performance of the so-called DAISY (161) in the more challenging SLT-I and SLT-II verocytotoxicity-neutralization assays was not... 

The deprotected lactosides were evaluated as inhibitors against lectin binding in a solid-phase inhibition assay with immobilized ASF on the surface of microtiter plate wells, mimicking cell-surface presentation, while mammalian galectins-1, -3, and -5 were in solution. Strong multivalency effects and selectivity were observed for the... 

The deprotected glycodendrimers 542 were tested using a newly developed ELIS A-based inhibition assay for their ability to inhibit the binding of recombinant type-1 fimbriated E. coli (FimH) to a monolayer of T24 cell lines derived from human... 

Wittekind E, Werner M, Reinicke A, Herbert A, Hansen P (1996) A microtiter-plate urease inhibition assay-sensitive rapid and cost-effective screening for heavy metals in water. Environ Technol 17 597-603... 

One of the most popular bioassay for interferons is termed the cytopathic effect inhibition assay . This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease. 

Several higher throughput in vitro assays may be used to assess various DMPK properties of NCEs. One common parameter is that HPLC/MS/MS is the method of choice for the analytical step.11 17 26 These higher throughput assays include the Caco-2 assay, p450 enzyme inhibition assay, and in vitro stability assay. Each assay has different requirements and solutions and they will be described individually. 

Di, L. et al. 2006. Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC/ S. Int. J. Pharmaceut. http //dx.doi.org/10.1016/j.ijpharm.2006.10.039. 

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