Positive control inhibitors

Positive control inhibitors for each of the major CYP enzymes should also be included to further demonstrate that the test system is performing as expected. The direct-acting inhibitors used in our laboratory are summarized in Table 7, along with the IC50 values determined during assay validation and a comparison with literature values. It is worth noting that the positive control inhibitors used for CYP inhibition studies need not necessarily be CYP-selective inhibitors, in contrast to those used for reaction phenotyping, which should be CYP-selective inhibitors. 

In the second example, two primary, in vitro serine protease assays with identical /Miitroaniline-based chromogenic (A405) readouts were either assayed separately or multiplexed. Positive control inhibitors specific to each enzyme were added to the assay at an appropriate concentration to determine assay... 

An automation procedure for sample preparation and incubation has been described previously (Yao et ah, 2007). A working solution of HLM was prepared with phosphate buffer to form 0.1 to 0.25mg/mL for the assays. Metabolite standards were manually prepared with 50% acetonitrile in water as stock solution, and further diluted with HLM for quantitation of the metabolites formed. Individual probe substrates were prepared at designated concentrations around their values. Positive controls (inhibitors) or test compounds were prepared in DMSO (or 50% acetonitrile) as stock solution. 

In an empty small volume (20 pL), black, 384-well HTS plate, prepare a dilution series of a positive control inhibitor in DMSO for subsequent use in screening plates in a next step, pre-dilute the compound series in buffer if required (see Note 38). 

If possible, measure the emission spectrum of the kinase in the presence and absence of the positive control inhibitor to verify that the desired maxima have not shifted in the HTS format. For some kinases, we have observed as much as a 10 nm shift... 

The % binding to the kinase is a way to quantify the percent of acrylodan-labeled kinase in the well which has adopted the DFG-out conformation in response to the binding of a compound and provides a straightforward comparison to the positive control inhibitor. The % binding is calculated using the equation ... 

In experiments with water-soluble inhibitors, the subsample was stirred under nitrogen during post-addition of an aqueous solution of the inhibitor followed by an aqueous sodium nitrite solution. Aliquots were weighed into 1-oz ointment jars, covered with nitrogen, sealed, and stored at 37 for later replicate analyses. Preparation of the positive control subsample was identical except that water was added in place of inhibitor. 

Each inhibitor was added to the oil phase of each emulsion before heating and combining the water and oil phases. Therefore, positive controls containing no inhibitor could not be made from the same base emulsion. Instead, a separate emulsion batch run simultaneously was used as a positive control. 

Experimental Results. The inhibition of NDEIA formation after days at 37 forthree water-soluble and three oil-soluble inhibitors is displayed in Tables II and III, respectively. So that relative effectiveness could be compared, the concentration of each inhibitor was chosen to provide an inhibitor/nitrite mole ratio = 10, Each inhibitor was tested in two separate emulsion batches with two or more aliquots analyzed. The data shown for positive controls are from similar replicate experiments. 

Figure 5.3 A convenient scheme for performing an inhibitor titration in 96-well format. Four compounds (1-4) are assessed in duplicate at each of 11 inhibitor concentrations. The inhibitor concentrations follow a threefold serial dilution from a maximum concentration of 1000 (molarity units nM, LlM, etc.). The right most column of wells is reserved for control samples. In this illustration four of the wells of column 12 are used for zero inhibitior positive controls, and the other four are used to establish the assay background as negative controls. Negative controls could represent any sample for which one knows that the enzymatic reaction has be abrogated. For example, the negative control wells could contain all of the reaction mixture components except the enzyme. See Chapter 4 for other potential forms of negative controls.

In many clinical trials a positive control of a clinically established drug is often used for comparison purposes for example, a novel selective serotonin reuptake inhibitor (SSRI), may be compared with a more established tricyclic antidepressant, such as imipramine. The aim is to see whether the new SSRI is more efficacious or has fewer adverse side effects than the more established tricyclic (Chapter 12). In many such comparisons the new and older treatments are equally efficacious at relieving depression, but the newer drugs display fewer side effects this means that they are better tolerated by patients, so that they are more willing to continue taking the tablets. The high rates of compliance also mean that, in overall terms, newer drugs with fewer side effects tend to be more efficacious. 

A general flowchart is presented in Fig. 13.5B that we followed for identifying and sorting inhibitors of translation. Shown below is an in vitro translation protocol tailored for ten 96-well assay plates (800 compounds), which can be scaled up or down as required. Negative and positive controls are present in wells A1 to D1 and El to HI, respectively. Compounds are added to wells A2 to Hll. Column 12 is left blank and could be used for additional controls, if desired. 

Animals, usually rodents, are exposed to a test substance by an appropriate route (usually oral or intraperitoneal injection, other routes may be appropriate) followed by administration of bromodeoxyuridine (BrdU). At least five female and five male animals per experimental and control group should be used. For an initial assessment, one dose of the test substance may be used, the dose being the maximum tolerated dose or that producing some indication of toxicity as evidenced by animal morbidity (including death) or target cell toxicity. For determination of dose-response, at least three dose levels should be used. A compound known to produce SCE in vivo should be employed as the positive control. A spindle inhibitor (e.g., colchicine) is administered prior to sacrifice. After sacrifice, tissue is obtained and metaphase preparations made, stained, and scored for SCE. 

If an aDNA extract frails to PCR amplify it should be tested for the presence of PCR inhibitors. This test requires the availability of an authenticated aDNA sample to be used as a positive control.8 Set up side-by-side PCR reactions containing 1) the template suspected to contain inhibitors, to which is added a volume of the ancient positive control equivalent to that of the template, 2) only the template suspected to contain inhibitors and 3) only the positive ancient control. This side-by-side comparison will allow for the preclusion of PCR failure due to factors other than inhibition (e.g. the stochastic nature of PCR amplification). If the template spiked with the positive ancient control (reaction 1) permits its amplification, while the template suspected of containing inhibitors (reaction 2) fails to amplify, the template is likely free of inhibitors and, therefore, does not contain a sufficient amount of DNA for analysis. Alternatively, if the first PCR reaction fails to amplify, whereas the third reaction does amplify, the template is concluded to contain inhibitors. In this case, the silica extraction should be repeated, as described above, and PCR reattempted. Our studies have shown that as may as four repeat silica extractions may be required to sufficiently remove PCR inhibitor from DNA extracts, despite the inherent loss of DNA yield associated with each repetition of the silica extraction (5). 

The use of positive controls is optional, but the FDA has nevertheless developed a list of preferred and acceptable inhibitors for use in reaction phenotyping studies that can be applied to CYP inhibition... 

Table 7 Inhibition of CYP Enzymes in Pooled Human Liver Microsomes by Direct Inhibitors (Positive Controls)... 

Overall approach. Hepatitis B viral extracts from human subjects are incubated with radiolabelled nucleotides and an active inhibitor. Percent inhibition is calculated based on the amount of de novo viral nucleic acid synthesized with respect to lamivudine as a positive control and phosphate buffer saline (PBS) as a negative control. 

A separate positive control experiment was performed in which a known DNA polymerase inhibitor (3 pi of lamivudine at a concentration 3 mg/ml) was used instead of the tested inhibitor (3 pi). 

Fig. 12.5 Positive control by growth factors and negative control byTGF-p are juxtaposed. The negative signals are transmitted to Cdki-cyclin A by the inhibitor, p27...

Compound 14 completely inhibited the prenylation at a concentration of 100 pM, the same effect as was seen with compactin, a known inhibitor of the mevalonate pathway used as a positive control. Selective inhibitor 17 showed 70% inhibition relative to compactin. No cytotoxicity was observed for all compounds up to 100 pM [41]. 

As in the previous example, the known crystal structures of Ach E-inhibitor complexes were taken as positive control to test the usability of AutoDock. The same standard docking parameters were taken as for the analysis of the ER ligands [12]. Due to the flexibility and the few polar functional groups of the inhi-... 

A number of different competitive cell-free and cell-based binding assays under static and hydrodynamic flow conditions have been used to obtain affinity data for selectin ligands. In addition to the fact that different positive controls have been used, this makes a direct comparison of reported binding affinities difficult. Therefore, in this chapter, we quote relative affinities wherever possible (Section 16.4.3). Another problem that has a negative effect on assay reliabihty has been encountered in cases where acidic ion exchange resins are used in the final step of the antagonist synthesis [170]. Small amounts of polyanions released from the resin were found to be potent selectin inhibitors, especially for P-selectin. These polyanions are difficult to remove and are not detectable by routine analysis. As a result, published assay data for P-selectin antagonists should be considered with caution. 

If reactions are performed in order to test new topi inhibitors, run a reaction with 1 pL of a 100 pM solution of CPT (10 pM final concentration) as a positive control. 

---