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With such a high level of discussion around the reproducibility of experiments described in the peer-reviewed literature, it is becoming increasingly prudent to scrutinize the tools that we use to conduct our research. Selecting a “good” probe is key to generating reliable and reproducible data, and it is part of our mission at the SGC to make these probes available to the research community.
“Chemical probes” are well-characterized small molecules that potently and selectively act on their target protein to elicit precise actions in cells. This enables the link to be made between inhibition or activation of target proteins and phenotypes in cell-based assays.
Chemical probe criteria are used to define acceptable quality standards and differ from one functional family to the next. The SGC requires fulfillment of the criteria in below (Table 1) as a benchmark standard before a compound is classified as an official epigenetic probe.
|In Vitro Potency (IC50 or Kd)||< 100 nM|
|Selectivity within Family||> 30-fold|
|Cellular Activity (IC50 or EC50)||Significant on-target activity at 1 μM|
Table 1. Chemical probe criteria for Epigenetic target family proteins.
The following blog aims to provide brief notes and guidelines to aid researchers in selecting and using their chemical probes.
As a first step, we recommend using a web-based chemical probe database such as Chemical Probes Portal (CPP), Probe Miner (PM), Guide to Pharmacology, and Probes and Drugs. These sites allow researchers to conduct target-centric searches, and provide detailed information about available probes for those targets. We also recommend to follow-up with a search of PubMed, before making your final selection.
Figure 1. Workflow for selecting a chemical probe.
Consider first the appropriate concentration to use in your assay; the key factors here are the probe’s potency and selectivity profile.
A dose-response comparison of primary (biochemical) with functional and/or phenotypic (secondary) events can establish a causal relationship between the target and phenotype. The primary response can often be monitored by a biomarker (such as the cellular levels of a histone methyl mark deposited by an enzyme inhibited by the probe). Deconvoluting the secondary response is more complex because cell viability is often used as a readout. At the IC90 of a chemical probe, the target is expected to be inhibited by 90% (as measured by the biomarker). If there is no effect on cell viability below the IC90, then the probe is likely acting on-target (this may be confirmed by genetic knockdown of the target). If however, cell death occurs at concentrations significantly lower than the IC90 then the effect of the probe on cell viability could well be via an off-target mechanism.
The shape of the cell viability curve may also reveal off-target effects. Excessively steep or shallow slopes have been associated with polypharmacology, population response heterogeneity or nonspecific toxicity. Further, apparent functional effects can occur at high compound concentration (relative to the effect on the biomarker). In this scenario, using two positive controls (with different chemotypes) is desirable to understand the pharmacology.
For a comprehensive review on how to use SGC chemical probes please click here.
The SGC aims to catalyze research in new areas of human biology and drug discovery by focusing on less well-studied areas of the human genome, such as epigenetic targets. By making well-characterized chemical probes available to the research community, the SGC has enabled a significant research output worldwide. One approach to illustrate the impact that the SGC’s epigenetic chemical probes have had is to evaluate the number of corresponding citations. A summary of this information by target class and selected individual probes is given below (Figure 2). The scientific impact of the BET family, G9a/GLP methyltransferase and JMJD3/UTX/JARID1B demethylase chemical probes is distinct and indicates the keen and continued interest in these key targets.
Figure 2: Top - Number of citations for each probe grouped by target class. Bottom - Number of citations since initial probe publication. Note that BET bromodomain chemical probes (+)-JQ1 (2010) and PFI-1 (2013) are not included in the histogram.(+)-JQ1 accounts for 1799 of the 1945 citations for these two chemical probes.
The SGC has developed a number of first-in-class chemical probes: GSK-J4 for JMJD3/UTX/JARID1B; SGC-CBP30 and I-CBP112 for the bromodomain of CBP/p300; LP99 for BRD9 and BRD7; OF-1 for BRPF1/2/3; PFI-4 for BRPF1B; and OICR-9429 for the methylysine binding domain of WDR5. Further, AbbVie has donated the first potent and selective acetyltransferase chemical probe, A-485, for CBP/p300 through the SGC’s Donated Probes Program. The Consortium of academic and industry scientists continue to strive to provide best-in-class chemical probes. There are now more advanced chemical probes (based on on-target potency and/or having a chemotype-matched control) for some of these targets. For example: BI-9564 and TP-472 (and its control TP-472N) for BRD9 and BRD7; and GSK6853 (and its control GSK9311) for BRPF1B.
On this note, we will close with a preview of the final blog in this series. It will give a brief overview of epigenetics and the use of chemical probes in research outside of oncology.