Elsevier

Drug Discovery Today

Volume 11, Issues 17–18, September 2006, Pages 855-859
Drug Discovery Today

Review
Post Screen
Avoiding physicochemical artefacts in early ADME–Tox experiments

https://doi.org/10.1016/j.drudis.2006.07.012Get rights and content

Recent literature has highlighted the importance of fundamental physicochemical properties in HTS and ADME–Tox: high lipophilicity, low dimethyl sulfoxide solubility, low aqueous solubility, aggregation and nonspecific binding to proteins and phospholipids. Unless carefully controlled for, each of these artefacts can confound individual ADME–Tox results and their interpretation on aggregate. This article reviews the impact of these phenomena and suggests experimental strategies for minimizing the magnitude and frequency of these artefacts without compromising the essential speed of the experimental process. Many of the concepts translate directly to the HTS context.

Section snippets

Aggregation

In 2002, Shoichet's research group [7] published a controversial study indicating that many compounds in HTS collections actually form aggregates at screening concentrations in buffer, and that these aggregates can interact with enzyme targets to produce false-positive hits. The possibility of ‘promiscuous inhibitors’ within the screening collections of many pharmaceutical companies suggests that at the concentration of compound typically used for screening, aggregation might be a very common

Solubility in DMSO and buffer

Another seminal paper, from Popa-Burke et al. in late 2004 [9], reported an analysis of the purification and quantification of the compounds in a screening collection at Amphora. In their analysis, DMSO samples were prepared freshly from purified, dried-down solids, thus minimizing the impact of repeated freeze–thaw cycles, DMSO hygroscopicity, DMSO evaporation, and other sources of compound instability in DMSO. Despite this caution, two troubling points were observed:

  • 1.

    The concentration of

The significance of this observation will vary according to the construct of the assay

In some assays, assumptions of starting concentrations appear in the mathematical formulas that convert measurements to ADME–Tox parameters. In this case, the impact is quite substantial. An example of this could be the computation of an inhibition constant for CYP inhibition.

In other cases, where ratios to starting concentrations are used, it is likely that the error will mathematically cancel; however, the proportion of drug sample to biological materials and/or physical phases will be quite

Nonspecific binding

Probably less of a surprise, but similarly subtle, is the problem of nonspecific binding within the assay construct. Lipophilic compounds are known to bind nonspecifically to proteins and biological membranes as well as lipid layers, filter media and some microtiter plate plastics. When they do so, the free fraction of compound – the amount of compound available to participate in the phenomenon of the assay – is reduced, sometimes even greatly. In other cases, particularly with low-soluble

Operating models for minimizing artefacts

At first pass, it would appear that the only course of action that can be pursued, to avoid the high potential cost of these artefacts, would be to test every compound in a more thorough assay, such as the example provided by Giuliano et al. [11]. However, this type or more costly assay takes more time, physical resources, scientific effort and reagents. In short, per dollar of research spending many less data of this sort can be produced compared with assays that are popular in early ADME–Tox

Conclusions

Recent evidence suggests that considerable care needs to be taken when performing ADME–Tox and HTS assays in vitro, particularly with lipophilic compounds. Because compounds can form aggregates at screening concentrations, buffer concentrations are often considerably lower than expected and nonspecific binding to protein, phospholipid or container materials can significantly reduce the free fraction of compound available to participate in the assay as designed.

Common approaches to deal with

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