The Chemical Probes Portal turns ten. Use it!

Last week we highlighted a new tool to computationally predict whether a molecule might aggregate, thereby causing false positives. This doesn’t necessarily mean the molecules are bad (after all, some approved drugs aggregate), but it’s all too easy to screen molecules under inappropriate conditions. This brings up the topic of chemical probes, and as it happens the Chemical Probes Portal turns ten years old this year, as celebrated in a Cancer Cell Commentary by Susanne Müller, Domenico Sanfelice, and Paul Workman and a blog post by Ben Kolbington at the Institute of Cancer Research.

 

We first wrote about the Chemical Probes Portal in July 2015, when it contained just 7 compounds. When we returned in 2023 it contained more than 500 compounds, and by the end of last year the number was up to 803. As of today it lists 1174 probes for 622 targets. Nearly a third of the probes also have chemically related inactive controls. These seem like large numbers, but the the human genome conservatively encodes for some 20,000 proteins, and the ambitious Target 2035 initiative seeks chemical probes for all of them.

 

The new paper emphasizes that the standards are in some ways higher for chemical probes than for approved drugs: “whereas probes principally require a high degree of selectivity, drugs need ‘only’ to be safe and effective and may often hit several targets.” Dimethyl fumarate comes to mind as a highly promiscuous covalent modifier that is nonetheless a useful drug for multiple sclerosis and psoriasis.

 

Even when a compound hits a target of interest, that doesn’t mean any biological effects observed are due to the target, particularly when the readout is cell death. The researchers note that TH588 was originally reported as a potent inhibitor of MTH1, but it actually kills cancer cells by binding to tubulin, a fact not always mentioned by chemical suppliers. Another study found that ten clinical compounds were still active in cells even when their putative target was knocked out using CRISPR.

 

The tone of the Commentary is pragmatic, emphasizing that for new or difficult targets, it may be difficult to find good chemical probes. For example, LY294002 is mentioned as a “pathfinder tool” that was useful to explore the biology around the PI3 kinase family but has now been superseded by more selective molecules.

 

Unfortunately, not everyone seems to have gotten the message. Curcumin, which as we noted can aggregate, form nonselective covalent adducts, fluoresce, and generate reactive oxygen species, appears in >2600 PubMed publications – just in the past year. What a waste.

 

If you’re exploring the biology of a target, please check the Portal to see whether there are good probes. If you’re reading (or reviewing!) a paper that reports small molecule studies, please check to see whether the probe has been assessed - especially to see if it shows up as one of more than 250 Unsuitables. And if you’re interested in participating, please consider reviewing or even hosting a Probe Hackathon.

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.

The Chemical Probes Portal at Eight

Back in 2015, Practical Fragments highlighted a new resource calling itself “The Chemical Probes Portal.” At the time it included just seven probes, and my post concluded, “I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments.”

 

Well, take off it has, as illustrated by a new (open access) paper in Nucleic Acids Res. by Susanne Müller (Goethe University Frankfurt), Bissan Al-Lazikani (MD Anderson Cancer Center), Paul Workman (Institute of Cancer Research), and collaborators.

 

The paper notes that “the widespread use of small molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behavior, has resulted in many erroneous conclusions in the biomedical literature.” As an antidote, the Portal is an “expert review-based public resource to empower chemical probe assessment, selection, and use.”

 

Any scientist can suggest a potential probe, and these are then internally reviewed and curated. Assuming enough public information is available about the molecule, probes are then sent to three members of a Scientific Expert Review Panel for further vetting. Reviewers rate probes from one to four stars for use in cellular and/or animal models and recommend relevant concentration ranges. Importantly, reviewers can also include comments to highlight off-targets, lack of certain data, oral bioavailability, or anything else.

 

From a mere seven probes in 2015 the Portal has grown to include more than 500 molecules covering more than 400 protein targets in about 100 protein families. About two thirds of the probes have three or more stars, meaning they are recommended. The Portal is very easy to use and can be searched by probe or protein. Laudably, all the data can also be easily downloaded in bulk.

 

In addition to the chemical probes, the Portal also contains around 250 “Historical Compounds” that have been described in the literature but “are not recommended to be used to study the function of specific proteins as they are seriously flawed.” These include molecules such as gossypol, a known aggregator that has been reported as an inhibitor of multiple proteins, and curcumin. If you see a molecule used as a probe in the literature, it’s worth checking to see whether it shows up in the Portal.

 

The Chemical Probes Portal features heavily in a Conversation between Cheryl Arrowsmith (Structural Genomics Consortium) and Paul Workman published (open access) last year in Nat. Commun. The researchers concisely define chemical probes as “small-molecule modulators to interrogate the functions of their target proteins, as opposed to protein location, or other physical properties.” Importantly, they differentiate chemical probes from drugs. “Drugs don’t necessarily need to be as selective as high-quality chemical probes. They just need to get the job done on the disease and be safe to use. In fact, many drugs act on multiple targets as part of their therapeutic mechanism.” I have frequently heard people make comments such as, “this is just a probe, not a drug,” but a good probe should actually be more selective than many drugs.

 

That said, you do want a drug to actually hit the target of interest. The researchers highlight iniparib, a putative PARP inhibitor that made it all the way to phase 3 clinical trials for breast cancer and was tested in >2500 cancer patients. It failed. Moreover, that failure cast a pall over the field which likely delayed the development of actual PARP inhibitor drugs.

 

The researchers also discuss aggregators, which are still being reported uncritically in the literature, along with PAINS. “Such compounds should never be considered further or used as chemical probes. They should be excluded from compound libraries. Yet many are sold by commercial vendors as chemical probes and widely used.”

 

This statement raised the hackles of Pete Kenny. In a recently published critique, he states: “it is asserted in the conversation that commercial vendors are selling compounds as chemical probes that are unfit for purpose and I strongly recommend that anybody making such assertions should carefully examine the supporting evidence.”

 

Dear reader, please try the following experiment. Enter “iniparib supplier” in your favorite search engine and see what comes up. For me, the first 10 results include several that describe it as a PARP inhibitor. I won’t link to them here because I don’t want to encourage traffic to their sites. (This is also part of the reason Practical Fragments has discontinued PAINS shaming, as it only increases the profile of sloppy or harmful papers.)

 

Pete goes on to write: “I would strongly advise against making statements that a compound is unfit for use as a chemical probe unless the assertion is supported by measured data in the public domain for the compound in question.”

 

Frankly, I don’t understand Pete’s position, which I parodied here. Life is short and biology is complicated, so why waste time with dirty or inadequately characterized reagents? For me, everything is an artifact until proven otherwise. And the Chemical Probes Portal goes a long way towards demonstrating whether a particular probe is fit for purpose.

ATAD2 Again...Now with a good tool.

Epigenetics is big.  We keep on beating that drum.  Just to prove it, today's paper is on a target we have talked about before: ATAD2.  That previous paper was unsatisfying: leading to my summary: "if you throw enough fragments at a target you can find a few that bind."  Today's entry  from GSK has produced the first micromolar inhibitors of ATAD2.  

As noted previously, ATAD2 is "undruggable" or at least VERY difficult to find chemical matter against.  To add to the difficulty,  the BET activity needs to be minimized.  With that in mind, they set a high threshold of activity (pIC50 greater than7) and 100 fold selectivity against BRD4 (a representative BET domain).  The ATAD2 site is more polar and flexible than BET.  The authors felt that this would be exploitable to create selective molecules.  To address ATAD2 they started with Ac-K mimics from previous BET work.  They supplemented this with diverse cores not represented.  One such array (which I read as libraries, somebody correct me if I am wrong) was based on the cpd 1,

Cpd 1. 

which is similar to the chemotypes discussed last year.   A crystal structure of 1 was solved, confirming that it bound as expected.  

Additional arrays were made around this core and tested in a TR-FRET assay.  30,000 compounds gave a 0.25% hit rate.  Confirmation was performed by HSQC NMR.  A subset of compounds interacted at the Ac-K site based upon comparison to compounds with known binding modes.  In this case, the peak that shifted upon binding were the same.  I would like to know if this was by visual inspection of spectra or if it was accomplished using PCA, or similar method.  It probably doesn't matter, but intrigues the NMR jock in me.

In rounds of medchem and X-ray confirmation, they were able to drive the potency against ATAD2 to the single digit micromolar.  The ligand efficiencies were maintained right around 0.30. Compound 57 (R=4-Me) and 60 (R=4-OMe) had the "best balance of ATAD2 and BET activity".  These compounds were also active in a cell-based assay known to be sensitive to BET inhibitors.  However, there is no selectivity.  ATAD2/BET pIC50 for 57 was 1.1 and 60 was 1.0. So, despite the selectivity threshold they developed, these compounds are not selective.  Despite that, I think this paper shows that the aphorism Undruggable =Undone is true.

Introducing the Chemical Probes Portal

Chemical probes can be incredibly powerful reagents for understanding biology. A potent, selective, and cell-active modulator of a specific protein can be invaluable for figuring out what that protein actually does. Fragment-based methods can be effective at identifying these tool compounds, as we've described here and here.

Unfortunately, good chemical probes are difficult to discover, and scientists are left struggling with suboptimal reagents that hit multiple targets, often through pathological mechanisms. This leads to "pollution of the scientific literature," in Jonathan Baell's memorable phrasing. Despite our occasional PAINS Shaming, high-profile articles in C&EN and Nature, and even a dedicated blog, the problem continues. What is to be done?

Yesterday, a team of 53 authors from 46 academic and industrial organizations published a Commentary in Nature Chemical Biology entitled "The promise and peril of chemical probes" (see here for excellent coverage in Nature, here for Science's take, and here for In the Pipeline). This provides a good working definition for a chemical probe. According to the Structural Genomics Consortium, a chemical probe for epigenetics targets must have:

• Potency < 100 nM against the desired target
• >30-fold selectivity vs related targets
• On-target cell activity < 1 µM

It should also be profiled against a larger panel of potential off-targets, and a related inactive compound (such as a stereoisomer) should be available as a control.

After discussing examples of high-quality probes, the researchers turn their attention to what they term – rather charitably – "probes of lesser value:"

The continued use of these probes poses a major problem: tens of thousands of publications each year use them to generate research of suspect conclusions, at great cost to the taxpayer and other funders, to scientific careers and to the reliability of the scientific literature.

The authors then go on to describe best-practices. For example, even high-quality probes can give spurious results when used at high concentrations. As Paracelsus recognized five centuries ago, the dose makes the poison.

All of this is important, but as the authors acknowledge, it's been said before. What really differentiates the Commentary is the simultaneous launch of a companion web site, the Chemical Probes Portal. Its creators hope that this will lead to vigorous community discussion around questions such as:

Is there a probe for my target protein?

Which ones should I use?

How should I use this probe properly?

Is this probe suitable for use in animal models?

Currently the Portal lists just seven probes with links to references and descriptions of selectivity, solubility, and the like. All of these are “good probes,” but hopefully this will expand: the paper itself discusses the shortcomings of molecules such as staurosporine, chaetocin, obatoclax, and gossypol, and including them in the portal with detailed warnings would be valuable for the scientific community.

I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments. Practical Fragments will check back in a year or so to see how the site is doing. In the meantime, probe cautiously!

With the proper tool, I could move the world

As noted before, bromodomains are a "hot" area of drug discovery.  Dan mentioned last year that PFI-1 was being released as a tool compound by the SGC.  In this paper, Fish et al. describe its discovery (Supplemental Information here).  They started their discovery with potential fragment-sized acetyl-lysine mimics (DMSO need not apply!), like others have described.  In particular, 3,4-dihydro-3-methyl-2(1H)-quinazolinones like Cpd 7 and its bromoequivalent.  These two compounds had sub-30uM potency and thus LE>0.45.  The efforts of Conway et al. and Chung et al. were highly instructive to the Pfizer group.  Crystallography was a key driver of confirming the binding modes seen by Conway are possible and that the quinazolinones are a viable acetyl-lysine mimic. 

The crystallography pointed out that the bromine is pointing towards solvent and thus the appropriate place to start doing chemistry.  Based upon the structures, a "bent" substituent at the 6 position appeared to be promising; sulfonamides were chosen for this role.    Compounds 9 and 11 were also noted as attractive, novel compounds in their own right.  These were used for very limited library construction.  The compounds derived from 9 were profiled first.  While better than the parent bromide, subsequent structural analysis showed that they were not making good interactions with the sites intended (WPF shelf).  The sulfonamides derived from 11 on the other hand showed significantly improved activity.  The SAR was relatively insensitive to the substitution of the aryl group, due to the optimized placement on the shelf and the reversed sulfonamide.  

PFI-1 has 0.22uM activity against BRD4 and it was nominated as the probe molecule.  They further investigated its binding via X-ray.  They also looked at it in a much broader array of assays: broader pharmacological selectivity, a cell-based inflammatory end-point assay, and rodent pharmacokinetics.  It had < 50% inhibition against 15 targets at 10uM (GPCR, ion channels, enzymes) and < 20% inhibition against 50 kinases. It fits the criteria for a good probe.

As the authors state, it was designed in a little over 250 molecules from an efficient fragment starting point covering only two design cycles.  I think this is an excellent example of probe design/discovery.