Probing the probes: fitness factors for small molecule tools

Abstract

Chemical probes for interrogating biological processes are of considerable current interest. Cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. To be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. Although recognizing that such guidelines may be valuable, we caution against overly restrictive rules that may stifle innovation in favor of a "fit-for-purpose" approach. Reviewing the literature and providing examples from the cancer field, we recommend a series of "fitness factors" to be considered when assessing chemical probes. We hope this will encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data, thus increasing understanding of the particular biological area, to the benefit of basic research and drug discovery.

Figure 1

Fitness Factors for Chemical Probes Grouped into Four Distinct Areas Chemical properties, biological potency, biological selectivity, and context of use (A), that encompass suggested criteria for evaluating the suitability of chemical probe compounds for exploratory biology (see Baell and Holloway, 2010; Cohen, 2009; Edwards et al., 2009; Frye, 2010; Inglese et al., 2007; Kodadek, 2010; McGovern et al., 2003; Oprea et al., 2007; Rishton, 2003). Threshold values that have been suggested in the literature are tabulated for each of the criteria (B), and a comparison of these properties for drugs, leads, and probes can be found in Table 1. We suggest that although not all probes can, or need to, reach these thresholds in every case, consideration of the criteria will allow a robust assessment of whether the probe is fit-for-purpose, and foster an appreciation of the risk carried forward if significant criteria are not met.

Figure 1

Fitness Factors for Chemical Probes Grouped into Four Distinct Areas Chemical properties, biological potency, biological selectivity, and context of use (A), that encompass suggested criteria for evaluating the suitability of chemical probe compounds for exploratory biology (see Baell and Holloway, 2010; Cohen, 2009; Edwards et al., 2009; Frye, 2010; Inglese et al., 2007; Kodadek, 2010; McGovern et al., 2003; Oprea et al., 2007; Rishton, 2003). Threshold values that have been suggested in the literature are tabulated for each of the criteria (B), and a comparison of these properties for drugs, leads, and probes can be found in Table 1. We suggest that although not all probes can, or need to, reach these thresholds in every case, consideration of the criteria will allow a robust assessment of whether the probe is fit-for-purpose, and foster an appreciation of the risk carried forward if significant criteria are not met.

Figure 2

Fitness Factor Liabilities for Selected Early Chemical Probe Inhibitors of Protein Kinases, the PI3K Family of Lipid Kinases and the HSP90 Molecular Chaperone Important fitness factors that have been improved significantly in subsequent chemical probes and clinical agents acting on these targets have been flagged. The symbols refer to the fitness factors listed in Figure 1, and the evolution of the probes to remove the liabilities shown is discussed in detail in the respective sections of the text.

Figure 3

Structures of Selected Protein Kinase Chemical Probes Discussed in Detail in the Text with Their Main Proposed Targets Indicated

Figure 4

Evolution of Increasingly Selective PKC Inhibitors Based on Staurosporine Percentage inhibitions for UCN-01 and ruboxistaurin were measured at 0.01 μM and 0.1 μM concentrations, respectively, for the same panel of 69 kinases (Bain et al., 2007). IC₅₀ values were determined for sotrastaurin in a panel of 32 kinases (Wagner et al., 2009). The cluster of potent activities for sotrastaurin represents PKC isoform inhibition. All three compounds inhibit several PKC isoforms with IC₅₀ 0.001–0.01 μM. Kinase dendrogram (Manning et al., 2002) reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Figure 5

Structures of Selected PI3 Kinase and HSP90 Chemical Probes Discussed in Detail in the Text with Their Main Proposed Targets Indicated