Best Practices for Chemical Probes
The recent re-launch of the Chemical Probes Portal, a community-led resource for chemical probe information, is a convenient time for reviewing best practices.
Chemical probes are small molecules developed to selectively inhibit protein targets. These tool compounds have been enormously valuable in helping us understand target biology. Validating probes for their selectivity and activity is not trivial, but is critically important for accurate interpretation of experimental data. Here are a few tips and recommendations.
1. Know your selectivity (and be suspicious of what you don’t know!). If you see an effect in an assay, be critical and consider if this is truly due to the primary target. To test this, additional experiments are necessary. These could include testing additional chemical probes having the same primary target and showing that these are also active in your assay, or running orthogonal assays such as target knockdown experiments. Always consider other explanations for your experimental results. Chemical probes are never perfectly selective for their primary targets. Indeed, often selectivity has only been defined for other members of the target class, or a subset of members. Not infrequently, probes have been discovered to interact with targets outside the primary target class. For example, several different kinase inhibitors, including the PLK1 inhibitor BI-2536, the JAK2-FLT3 inhibitor TG-101348 and the PI3K inhibitor LY294002, were found to interact with members of the BET bromo-domain protein family. Also, many compounds due to their chemical nature can non-specifically alter processes such as membrane trafficking within the cell, confounding results from cell-based and in vivo assays.
2. Know your application. The rules for using chemical probes are different for biochemical versus cell-based experiments. For intracellular targets in cell-based assays, the probe must get into the cell and be sufficiently stable to locate and interact with the target. Chemical features that enable metabolic stability and cellular uptake are unrelated to the features involved in target engagement, and so do not necessarily correlate. Biochemical potency is not the same as cell-based assay potency, but cell-based potency is more important for predicting effects in vivo. Secondary or off-targets (known and unknown!) are more likely to contribute to activities in cell-based and in vivo assays, because these targets are more likely to be present in these systems. Using the right probe concentration is also an important issue. In biochemical assays this is easy to measure and control. However, for cell-based assays and in vivo studies, we don’t know how much is getting into the cell, or concentrations achieved in relevant tissues. As a general guideline for translating concentration effects between in vitro and in vivo systems, consider testing probes in vitro at concentrations 10-fold above the maximal in vivo exposure level (e.g. Cmax). Don’t forget that for most drugs in vivoexposures range 5-10 fold between individuals, so a 100-fold window is not unreasonable. If you are looking to estimate a therapeutic (or toxicity) window, it is best to start in vitro at a top concentration of 300-fold above the average animal or patient maximal exposure.
3. Controls, controls, controls. Controls are critical for chemical biology experiments. Although these are rarely available, a structurally related compound that does not interact with the target is a great control to have. More frequently, there will be multiple structurally diverse probes available and these should be tested. For some targets, there may be both agonists and antagonists available. It is also highly recommended to perform concentration-response testing to fully characterize probe effects (and don’t forget to run sufficient numbers of replicates to characterize your assay variability). Concentration-response information is highly helpful for probe characterization. We have observed that highly potent and selective probes tend to be active in cell-based assays over a wide concentration range. This feature (termed dose resistance) is characteristic of high quality chemical probes and differentiates the more useful probes from those of low quality, such as those described in Arrowsmith, et al., 2015. This feature also related to in vivo therapeutic window.
These are just a few personal recommendations. For more information on the challenge and value of chemical probes, see here, here and here.
And be sure to visit the Chemical Probes Portal for the latest recommendations and be a contributor!
