Spotlight Cite This: ACS Chem. Biol. 2018, 13, 1109−1110
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Transparent data sharing is one powerful way of evaluating a probe’s suitability. Probe Miner is an online public database that aggregates binding and bioactivity data for more than 1.8 million small molecules. The result is a large and free database which can be easily searched by molecule or target, which provides information on in vitro potency, cellular potency, selectivity, structure−activity relationships, inactive analogues, and a filter for pan-assay interference (PAINs) elements (http://probeminer.icr.ac.uk). This platform offers the first data-driven evaluation of chemical probes on this scale with great attention paid to a broadly accessible interface. In addition, it allows analysis of objective and quantitative metrics. It is also worth noting that the quality of data for a given probe is improved by its broad exploration by multiple groups, further encouraging wide dissemination of chemical reagents. A complementary resource is offered at ChemicalProbes.org, a database that utilizes a panel of highly qualified chemical biologists, medicinal chemists, and drug hunters to offer expertly curated information on tool compounds. While covering fewer molecules than Probe Miner; the ChemicalProbes.org approach provides high quality ratings of molecules, including whether they are suitable for use in cells or animals to inhibit the reported target. ChemicalProbes.org also puts great care into establishing an intuitive rating scale which takes little experience in chemistry or pharmacology to interpret. Recently, a consortium of pharmaceutical companies has made a public database of binding data and usage guidelines that covers a previously confidential set of inhibitors. In this database, Open Science Probes (http://www.sgc-ffm.unifrankfurt.de) usage recommendations, inactive analogues, and extensive validation data are disclosed for numerous tool compounds. This resources provides a new set of powerful tools for academic investigators.
hemical probes are powerful tools because they offer dose-dependent, selective, and temporal control over a protein’s function. Molecules such as rapamycin and bortezomib have revolutionized our knowledge of their biological target’s roles in health and disease. However, the opposite is also truea bad (e.g., nonselective) chemical probe can lead to confusing or contradictory findings. What makes a good chemical probe? Clearly, one needs to have a detailed understanding of the molecular target(s) of the molecule. While polypharmacology is a common mechanism for drugs and may even boost efficacy in many cases, the job of a chemical probe is to selectively perturb a single protein or family of proteins in a well-characterized way. At the very least, one should understand how many targets are being effected and at what concentration. One also needs to understand the effective concentration of the molecule needed to inhibit the target(s) and how much inhibitor is present in the biological model (e.g., cell, animal). In other words, at some concentration, most/all molecules will have off-target effects, so one needs to understand target engagement and its limits. In cells, this value will be a product of not only direct interaction of the molecule with its target but also of the compound’s permeability, metabolism, and transport. In mammalian models, first pass hepatic metabolism and other clearance pathways are also key considerations. Thus, in order to produce quality chemical probes, significant expertise in medicinal chemistry, biochemistry, cell biology, and pharmacology are required. This concept is expertly summarized by Stephen Frye’s commentary: “The art of the chemical probe” (2010) in which he outlines five principles for evaluating chemical probes paraphrased as follows: 1. Molecular prof iling is sufficient to relate in vitro potency and selectivity to in vivo activity. 2. Mechanism of action is clear, allowing biological activity to be attributed to the perturbation of the target of interest. 3. Identity of the active species is clearly and robustly relatable to the function in vitro and in vivo. 4. Utility as a probe has been shown through use as a tool compound to investigate a hypothesis about the target. 5. Availability of the compound is guaranteed for open use by the academic community. Frye further highlights that the typical lab does not have the resources or expertise to accomplish such efforts on its own. Often, a chemical probe must be produced, circulated, optimized, and evaluated by many groups before decisions on its utility can be reached or initial issues overcome. Further, each new biological system/model might add its own idiosyncrasy (e.g., cell wall in yeast), necessitating re-evaluation of the probe for each new application. We focus this commentary on updating the online resources available for evaluating chemical probes, which have appeared since Frye’s seminal description. We also want to highlight the new technologies, such as cellular thermal shift assays (CETSA) and CRISPR-mediated interference and activation (CRISPRi/a), which have emerged to improve evaluation and optimization of chemical probes. © 2018 American Chemical Society
Box 1. Resources for the advancement of chemical probe validation and availability Probe Miner is an open database which aggregates public binding and activity data to objectively and quantitatively evaluate chemical probes. • http://probeminer.icr.ac.uk/#/ ChemicalProbes.org is a collection of expert-curated probes with usage suggestions, limitations, and considerations clearly described. • http://www.chemicalprobes.org/. Open Science Probes is a recently started database of donated data on precompetitive compounds from industry which are of likely useful as chemical probes. • http://www.sgc-ffm.uni-frankfurt.de. Selectivity and target engagement are key concerns for a good chemical probe. However, validating these issues is technically Published: May 18, 2018 1109
DOI: 10.1021/acschembio.8b00390 ACS Chem. Biol. 2018, 13, 1109−1110
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dose-responsive stabilization of a protein implicates it as a likely target. Because the readout does not rely on protein function, CESTA is quite general.
challenging, and available methods, such as SEREP panels and kinase/GPCR screens, are expensive. This gap has spurred development of generalized target validation platforms. First, CRISPRi/a screens utilize a catalytically dead Cas9 (d-Cas9) coupled with either a transcriptional repressor KRAB domain (i) or transcriptional activators (a) in order to knock down or overexpress endogenous genes. A critical aspect of this platform is that it can be massively multiplexed, making genomewide screens feasible. Further, it is an improvement on previous large-scale shRNA screens because of increased selectivity and fewer off-target effects. In a typical CRISPRi/a experiment, cells stably expressing dCas9 are split into at least two populations (one compound-treated and one untreated) and transfected with a library of sgRNAs, each targeting the transcriptional start site of a gene. This library can represent a subset of the genome (e.g., when the approximate target class is known) or even the entire genome. The experimental population is treated with the candidate probe, and cells are passaged. Essentially, if knockdown/ overexpression of the gene provides either resistance or sensitivity to the compound, then it will alter the proportion of the cells that are in the population, relative to control. One key element of these screens is the choice of phenotype. For cell survival as a phenotype, cells are harvested at t0 and compared to tf for both treated and untreated types. Loss of a specific sgRNA abundance is detected by short-read next-generation sequencing of the sgRNAencoding DNA. The enrichment for a sgRNA is calculated relative to the average change in abundance of thousands of nontargeting guides. This approach is similar to synthetic lethal screens in yeast, but CRISPR technology has allowed a conceptually parallel approach in mammalian cells, for the first time. The list of sensitivity and resistance genes often includes the primary target, as well as off-targets and other components of the affected pathway. In addition to cell growth phenotypes, fluorescent reporters are being increasingly used, while next generation methods will likely need to incorporate additional phenotypic measures. Another technology which has provided a similarly broad applicability is DrugTargetSeqR, which utilizes the mutationprone and efflux pump deficient HCT116 cell line to conduct a directed evolution experiment in the presence of a candidate probe. Following cell passages in the presence of compound, the resulting population and parenteral population are deep sequenced. Upon comparison, mutations in the direct target(s) and related pathways are enriched. Reoccurring mutations are then progressed to a “nickase” Cas9 (nCas9)-mediated selection. Briefly, in a genetically stable cell line, nCas9which produces single-stranded DNA breakscan be directed toward each locus implicated in the HCT116 directed evolution with sgRNA. Intrinsically error-prone, homology-directed repair (HDR) will recognize the break and produce mutants at the desired locus. In the presence of a probe, this library will be selected for resistant mutants. With DrugTargetSeqR, genomewide mutational analysis yields candidate mutations which may provide resistance, and directed mutational library production gives information on which mutations are capable of independently conferring resistance. Finally, CESTA takes advantage of the stabilizing effect of a ligand when it binds to a target protein. In CESTA experiments, the candidate probe is added to lysate, and this lysate is heated. Denatured proteins are then separated as insoluble aggregates by centrifugation, and the supernatant is assayed for the presence of the target of interest and potential off-targets by Western blot. Alternatively, proteome-wide analyses can be conducted by mass spectrometry of the soluble fractions. In either case, a
Box 2. Suggested literature regarding chemical probe quality and validation Frye, S. V. (2010) The art of the chemical probe. Nat. Chem. Biol. 6, 159−161, DOI: 10.1038/nchembio.296. Antolin, A. A., Tym, J. E., Komanou, A., Collins, I., Workman, P., and Al-Lazikani, B. (2017) Objective, quantitative, data-driven assessment of chemical probes. Cell Chem. Biol. 25 (2), 194−205, DOI: 10.1016/j.chembiol.2017.11.004. Arrowsmith, C. H., Audia, J. E., Baell, J., et al. The promise and peril of chemical probes. Nat. Chem. Biol. (2015). 11, 536−541. doi: 10.1038/nchembio.1867. Baell, J. B., and Nissink, J. W. M. (2018) Seven year itch: Pan assay interference compounds (PAINS) in 2017−utility and limitations. ACS Chem. Biol. 13 (1), 36−44, DOI: 10.1021/acschembio.7b00903. Jost, M., and Weissman, J. S. (2018) CRISPR approaches to small molecule target identification. ACS Chem. Biol. 13 (2), 366−375, DOI: 10.1021/acschembio.7b00965. Kasap, C., Elemento, O., and Kapoor, T. M. (2014) DrugTargetSeqR: a genomics- and CRISPR-Cas9-based method to analyze drug targets. Nat. Chem. Biol. 10, 626− 628, DOI: 10.1038/nchembio.1551. Molina, D. M., Jafari, R., Ignatushchenko, M., et al. (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 6141 (341), 84−87, DOI: 10.1126/science.1233606. Bondeson, D. P., Mares, A., Smith, I. E. D., et al. (2015) Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611−617, DOI: 10.1038/ nchembio.1858. It is clear that a good chemical probe requires extensive work, often by multiple laboratories. To circumvent this timeline, multiple chemical biology approaches have emerged to more rapidly validate a target prior to the investment of significant medicinal chemistry efforts. For example, inducible degradation domains (e.g., degrons) can be appended to the target protein to gain chemical control over its function. This approach allows one to use a well-validated compound to stabilize the degron and explore the function of virtually any protein. More recently, proteolysis-targeted chimera (PROTAC) methods have also become available. Briefly, PROTAC uses a scaffolding bifunctional molecule with affinity for an E3 ubiquitin ligase and a protein of interest to bring these molecules into close proximity. This scaffolded proximity leads to the ubiquitylation and degradation of the protein of interest. Thus, PROTAC allows the rapid degradation of an unmodified protein, providing an alternative to degrons. In the eight years since Frye’s review, great strides have been made toward supplying online resources for choosing and evaluating chemical probes. Importantly, many of these resources are available to the nonspecialists, allowing informed decisions about which compound(s) would be valuable for high quality biological experiments. In addition, new technologies have been developed to assist in chemical probe evaluation and optimization. The job is not done, but there seems to be a growing realization of the importance of rigor, reproducibility, transparency, and high quality chemical probes. 1110
DOI: 10.1021/acschembio.8b00390 ACS Chem. Biol. 2018, 13, 1109−1110
