A rule of two for using chemical probes?

Earlier this year we highlighted the growth of the Chemical Probes Portal, a free website that profiles more than 500 small molecules targeting more than 400 proteins. Each chemical probe is evaluated by experts based on published literature and then scored for use in cells or in vivo. More than 300 chemical probes have received three or four stars and are thus recommended. But even a good probe can be misused, and this is the subject of a recent (open-access) Nat. Comm. paper from Adam McCluskey, Lenka Munoz, and colleagues at the University of Sydney and the University of Newcastle. (The paper has also been discussed by Paul Workman and Derek Lowe.)

 

The researchers chose eight probes targeting histone methyltransferases, a histone demethylase, a histone acetyltransferase, and several kinases. All but one of these probes had first been disclosed before 2015. A literature search revealed 662 papers that used these probes in cellular studies, ranging from 21 to 134 publications per probe.

 

Centuries ago the alchemist Paracelsus noted that everything is poisonous at high enough doses, and indeed even the best probes might hit dozens or hundreds of protein targets. For this reason the Chemical Probes Portal recommends maximum concentrations for cellular assays. The researchers examined whether papers exceeded these concentrations. The overall results were encouraging, with just 22% of papers exceeding recommended limits. However, there was considerable variation: for one chemical probe, 70% of papers exceeded the limit. (For this particular case, the maximum recommended cellular concentration was just 250 nM.)

 

Because chemical probes can have off-target activity even at recommended concentrations, best practices are to include a related but inactive control compound plus a second chemically differentiated probe. All but one of the eight probes chosen for analysis had orthogonal probes available, and five had inactive controls. So how frequently were these used? Unfortunately, 58% of papers did not use an orthogonal probe, and a whopping 92% of papers did not use available inactive control compounds. In fact, just 4% of the papers “used chemical probes within the recommended concentration range and included inactive compounds as well as orthogonal chemical probes.”

 

A wider analysis of nearly 15,000 papers that cited the 662 publications produced similar results, with 17% exceeding recommended concentrations, 59% not using differentiated chemical probes, and 83% not using inactive controls.

 

The researchers propose a “'rule of two': At least two chemical probes (either orthogonal target-engaging probes, and/or a pair of a chemical probe and matched target-inactive compound) to be employed at recommended concentrations in every study.” To encourage best practices, the paper provides a simple “Researchers’ Flowchart” to help investigators select probes and controls. And because science is self-regulated, they provide a five-item “Reviewers’ Checklist.” The paper also includes a nice list of links to other resources, including webinars and slide decks.

 

Overall I think following these guidelines would be beneficial, and the Reviewers’ Checklist in particular could be usefully incorporated into journal publication requirements.

 

Of course, the vast majority of protein targets don’t have even a single good chemical probe, let alone two or more. Which means that there are plenty of opportunities to identify new probes and make better use of those that already exist.