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Learning from PAINful lessons Daniel A. Erlanson* Carmot Therapeutics Inc., 409 Illinois Street, San Francisco, California 94158, United States ABSTRACT: Chemical probes are important both as tools to understand biology and as starting points for drug leads, but not every active molecule makes a good probe; many react nonspecifically with thiols. These promiscuous inhibitors are worse than useless because they can mislead researchers and muddy the literature. Understanding the mechanisms of such compounds can prevent scientists from following false hits down blind alleys.
It is better to know nothing than to know what ain’t so.
molecules proved to be legitimate: the remaining 99.8% turned out to be artifacts. Although this fraction may seem high, experienced screeners will probably not be surprised. What distinguishes this paper is that the researchers carefully characterized the mechanisms of these pathological molecules. Five different chemotypes were investigated (Figure 1, top). Many manifestations of these were quite potent, with IC50 values in the low micromolar and even high nanomolar range in the CPM assay, which assesses activity based on the amount of thiol−CPM adduct. However, the compounds had similar activity when tested with just the thiol-containing coenzyme A reaction product; in other words, the results were the same whether or not any Rtt109 enzyme was present. This suggested that the compounds were behaving as electrophilic thiol traps for coenzyme A, preventing it from reacting with CPM. The researchers incubated representative compounds with either coenzyme A or glutathione, another common biological thiol, and analyzed the reactions with ultraperformance liquid chromatography mass spectrometry (UPLC−MS). They found that all five chemotypes could react covalently with thiols. Specifically, a thiol can react with chemotype 1 through a Michael addition-elimination, displace the aromatic thiol of chemotype 2 through nucleophilic aromatic substitution, or open the 1,2,4-thiadiazole ring of chemotype 3. Chemotype 4 can spontaneously eliminate an aromatic thiol in slightly basic buffer to generate a reactive maleimide. And chemotype 5 (which had been previously recognized as a PAINS) turned out to be a “triple threat”: it can react directly with thiols, but it can also generate hydrogen peroxide, which can go on to oxidize thiols. Moreover, molecules containing chemotype 5 as well as their thiol adducts are unstable in solution and decompose to naphthoquinones, which can also react with thiols and generate hydrogen peroxide. For all five chemotypes, some representatives showed activity in orthogonal, antibody-based, or radiolabeled assays of histone acetylation. However, adding the reducing agent dithiothreitol (DTT) dramatically attenuated this effect, suggesting covalent modification of cysteine residues on the protein. This was confirmed using proteolytic digestion and mass spectrometry. Although Rtt109 has several cysteine residues, none are known to be important for catalysis, so the loss in activity likely reflects
Josh Billings
You have spent the past several years studying an important biological pathway, convinced that you have found the key to vanquishing a serious disease. No one has reported small molecule modulators of your target, so you decide to find one yourself: perhaps you work with a screening center, or you set up a screen in your own lab. And you find hits! You file a provisional patent application and write up a manuscript for publication, hoping to share your hard-won knowledge with a world in need of new drugs. Happy stories like this are how science is supposed to work. But all too often the hits identified are artifacts, molecules so promiscuous that they will never become a drug. At best, the resulting publications are ignored. At worst, they lead to confusion as other researchers use the supposed chemical probes to misunderstand biology. Although these molecules may show activity in cell or even animal models, they inhibit so many other pathways that it is impossible to link activity to the target of interest. In this issue, Michael Walters and collaborators at the University of Minnesota, the Mayo Clinic, and AstraZeneca offer new defenses against this danger.1 In fact, the opening story describes the beginning of Walters’ journey. His lab was interested in the histone acetyltransferase Rtt109 as a potential antifungal target. To look for inhibitors, they developed a high-throughput screen (HTS) that detects a thiol-containing byproduct of the enzymatic reaction. This type of assay, in which the molecule N-[4-(7-diethylamino-4methylcoumarin-3-yl)phenyl]maleimide (CPM) reacts with a thiol to form a fluorescent adduct, is commonly used, particularly for enzymes that use the cofactor acetyl coenzyme A. After screening ∼225 000 compounds, they obtained ∼1500 apparent hits. Pan-assay interference compounds, or PAINS, are a particularly pernicious class of assay artifacts.2 PAINS comprise a staggering variety of different chemical structures; their common feature is that they show up as hits in many different assays and can rarely be optimized to specific inhibitors. The researchers were aware of these types of compounds and had used computational filters to weed out molecules that contained PAINS substructures. Nonetheless, closer inspection of the 1500 hits revealed that many were similar to PAINS but had not been caught by the cheminformatic filters. After testing these hits in orthogonal assays and counterscreens, only three © 2015 American Chemical Society
Received: February 19, 2015 Published: February 24, 2015 2088
DOI: 10.1021/acs.jmedchem.5b00294 J. Med. Chem. 2015, 58, 2088−2090
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Figure 1. (Top) New PAINS. These substructures have been shown to react readily with thiols and thus should be treated with extreme caution when they appear in screening hits or published probes. (Bottom) Misleading probes. These molecules are commercially available and have been reported in the literature as active against various targets. However, according to the work reported by Dahlin et al.,1 they are not specific for their targets, thus casting doubt on biological conclusions based on them.
Table 1. Methods To Weed Out Nonselective Thiol-Reactive Moleculesa cheminformatics and computational • Substrate filters (PAINS, REOS) • Computational calculations (FMO) • Literature/patent searches
counterscreens • Target sensitivity to reactive compounds • Activity versus unrelated targets • Activity with and without DTT
mechanistic experiments • Compound−target dilution
redox activity • Redox assays (HRP-PR, resazurin, cysteine proteases)
• Activity with and without dialysis • Time dependence
small molecule thiol probes
protein probes
• For example, cysteamine, GSH, CoA, MSTI
• Peptides
• Direct detection (HPLC−MS)
• ALARM NMR/MS
• Fluorescence (MSTI, CPM-CoA competition)
• Compound reactivity with protein target (protein MS)
• Screening databases (PubChem) • Fundamental principles of medicinal chemistry a
See Dahlin et al.1 for full details and references.
In fact, some of these molecules are even sold commercially as chemical probes, as shown at the bottom of Figure 1. Any research based on reported biological activity of these molecules should be treated cautiously, at best. Importantly, the authors are not impugning covalent drugs per se. Indeed, many approved drugs react covalently with biological molecules, which can lead to longer target engagement and lower dosing.3 The key difference is that covalent drugs are mostly specific for their targets, whereas the molecules reported here will react indiscriminately with exposed cysteine residues or with assay components. This paper is important for several reasons. First, it reveals new PAINS that should be avoided. Second, it demonstrates the insidious nature of PAINS: in some cases more than two dozen representatives of a given chemotype had widely varying activities, and these SIRs could be misinterpreted as SARs. Third, it emphasizes that computational filters are inevitably inadequate and that nothing can substitute for thorough mechanistic analysis to (in)validate a molecule as a selective mechanistic probe of biological function. To this end, the paper provides an armamentarium of tests and controls to eliminate thiol-reactive molecules, summarized in Table 1. Reproducibility in science has recently received considerable attention. However, PAINS are arguably even more damaging
general denaturation. Additional experiments using MS and nuclear magnetic resonance (NMR) revealed that representative compounds also modify other, unrelated proteins, in some cases denaturing them. Thus, these molecules are likely to interfere with a wide variety of assays, not just those that use thiol-reactive reagents. Finally, the researchers interrogated public (PubChem) and private (AstraZeneca) screening data to determine how often these substructures showed up in active molecules. Not surprisingly they were overrepresented, in some cases coming up as hits in more than a fifth of the hundreds of screens conducted. PAINS are particularly problematic because not all representatives of a given chemotype are active in any given assay. This variability, which is due to reactivity and solubility differences, has been dubbed structure−interference relationships (SIRs) and can easily be mistaken for the structure− activity relationships (SARs) sought by medicinal chemists. Unlike SAR, SIR does not depend on specific recognition by a biological target. Sadly, confusion between SIR and SAR is frequent, and representatives of each of the five chemotypes have been reported as hits in the literature. The Supporting Information contains dozens of references reporting examples, representing enormous wasted effort by hundreds of scientists. 2089
DOI: 10.1021/acs.jmedchem.5b00294 J. Med. Chem. 2015, 58, 2088−2090
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than irreproducible results: they tend to be highly reproducible but for the wrong reasons. While they will repeatedly inhibit a given assay, they will also inhibit many other targets known and unknown, so testing them in cells or more complex assays will produce uninterpretable (though too often interpreted!) results. The story at the beginning of this Viewpoint has a surprise but happy ending: although the researchers found very few inhibitors of Rtt109, they did discover new classes of PAINS which they are now sharing with the world. If you encounter molecules containing these substructures, whether in a screen, a manuscript you are reviewing or editing, or a publication, you need to ask tough questions. And of course this paper only considers PAINS; many other types of artifact await the unwary.4
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (415) 978-2159.
ACKNOWLEDGMENTS I thank Monya Baker for a careful reading of this Viewpoint. REFERENCES
(1) Dahlin, J.; Nissink, W.; Strasser, J. M.; Francis, S.; Higgins, L.; Zhou, H.; Zhang, Z.; Walters, M. A. PAINS in the assay: chemical mechanisms of assay interference and promiscuous enzymatic inhibition observed during a sulfhydryl-scavenging HTS. J. Med. Chem. 2015, DOI: 10.1021/jm5019093. (2) (a) Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53 (7), 2719−2740. (b) Baell, J.; Walters, M. A. Chemistry: Chemical con artists foil drug discovery. Nature 2014, 513 (7519), 481−483. (3) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307−317. (4) Davis, B. J.; Erlanson, D. A. Learning from our mistakes: the “unknown knowns” in fragment screening. Bioorg. Med. Chem. Lett. 2013, 23 (10), 2844−2852.
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DOI: 10.1021/acs.jmedchem.5b00294 J. Med. Chem. 2015, 58, 2088−2090
