High Throughput Screening

The development of robust and sensitive assays for finding inhibitors or allosteric modulators of ligand binding and/or receptor activity is critical to screening that will allow the discovery of new leads. Traditional screening methods used membrane preparations and specific, radio-labeled ligands. Compounds that competed with a labeled ligand were identified after the separation of the unbound radioactivity by filtration. Even though receptor-radioligand binding screens are robust, they do not provide information on the intrinsic activities of a compound; all active compounds are identified only as inhibitors of the ligand-receptor interaction in these primary screens.

Biochemical or cell-based functional assays constitute information-rich platforms capable of distinguishing agonists, antagonists, partial agonists, inverse agonists, allosteric modulators, and allosteric enhancers (potentiators). They can identify novel leads with desirable properties for a therapeutic target. Quality control is an important consideration when implementing an HTS campaign. The robustness of the HTS assay is measured by the Z' factor determined by analyzing multiple wells of total binding and nonspecific binding.11 The Z' factor is calculated using the following equation:

z' = 1 - (3os + 3 oc)/ms - mc where ss = total binding mean standard deviation; oC = nonspecific binding mean standard deviation; ms = mean of total binding; and mc = mean of nonspecific binding.

The Z' factor determines the inherent noise of the assay. If the Z' factor is good (>0.5), further development will be conducted to ensure a robust screen:

1. The affinity of the known benchmark compounds will be determined and compared with the published values.

2. As the test compounds are solubilized in dimethyl sulfoxide (DMSO), the sensitivity of the assay to DMSO will be ascertained in the presence of different concentrations of DMSO.

3. Drug concentration to be used in the final screening will be determined by analysis of scatter plots produced with varying compound concentrations; this includes determining the Z factor.

The Z factor, similar to the Z' factor, is a screening window coefficient calculated by analyzing wells containing drugs and wells defined for nonspecific binding. The screening concentration is chosen so that the Z factor produced is in the range of an excellent assay and the final DMSO concentration in the well does not negatively impact the quality of the assay. Usually a Z factor of 0.5 to 1 is considered an excellent assay; 0 to 0.5 is a "doable" assay.

After the primary screen, the inhibitory wells from the primary screens are retested and Ic50 and Ec50 values are determined after performing a concentration-dependent titration. Screening libraries are usually maintained as DMSO-solubilized liquid stocks and often stored for convenience at room temperature. Several recent studies indicate that these storage conditions produce precipitation and partial degradation of compounds.12-14 In light of these studies, many screening laboratories consider stability as well as throughput and are converting their stores into inert low temperature facilities. It is always advisable to reconfirm the activities of the powder sources and confirm the structures of the compounds.

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