Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

Abstract

Small-molecule chemical probes or tools have become progressively more important in recent years as valuable reagents to investigate fundamental biological mechanisms and processes causing disease, including cancer. Chemical probes have also achieved greater prominence alongside complementary biological reagents for target validation in drug discovery. However, there is evidence of widespread continuing misuse and promulgation of poor-quality and insufficiently selective chemical probes, perpetuating a worrisome and misleading pollution of the scientific literature. We discuss current challenges with the selection and use of chemical probes, and suggest how biologists can and should be more discriminating in the probes they employ.

Keywords: PAINS; Pan-Assay Interference Compounds; biochemical selectivity; chemical probe; chemical reactivity; chemical tool; ligand promiscuity; lipophilicity; pharmacological audit trail; pharmacophore crossing; target validation.

Figure 1

Fragments to Fabs: an Affinity-Selectivity Spectrum Increasing molecular weight increases the likelihood of specificity. Careful chemical optimization and biological testing must be carried out to minimize the risk of off-target effects in chemical probes.

Figure 2

Dos and Don'ts of Chemical Probes

Figure 3

MTH1 and Histone Demethylase Chemical Probes (A) MTH1 ligands TH287 and TH588 with lipophilic moiety highlighted in lilac, S-crizotinib, and characterized chemical probes 1 and 2. (B) Histone demethylase inhibitor GSK-J1 with the highly ionized and poorly cell-penetrant carboxylic acid moiety highlighted in yellow and its corresponding cell-penetrant ethyl ester GSK-J4 (ester moiety highlighted in green).

Figure 4

Factors Likely to Influence Promiscuity in a Claimed Chemical Probe (1) Chemical reactivity and instability (a chemical structure that is reactive or unstable in the medium of a biological assay); (2) lipophilicity (the propensity for a small molecule to leave the aqueous environment and bind to proteins irrespective of the protein structure or sequence); (3) chemical substructures associated with assay interference (Pan-Assay Interference Compounds, PAINS, see text). The overlap in the Venn diagram indicates that more than one undesirable feature can occur in a given chemical structure which magnifies the likelihood of problems occurring in biological use.

Figure 5

Thio-2, Lowering Lipophilicity and Pharmacophore Crossing (A) Chemical structure of Thio-2 with the reactive metabolite precursor highlighted in orange. (B) Lowering the calculated lipophilicity (cLogP) of the PAK1 inhibitor chemical tool G-5555 by 100-fold compared with starting compound FRAX1036 by removal of the lipophilic side chain (lilac) bearing a highly basic nitrogen and addition of a tolerated polar side chain (green) bearing a weakly basic nitrogen. (C) Pharmacophore crossing: the PLK1 kinase-binding motif of BI2536 is highlighted in purple and the BRD4 Asn140-binding motif is shaded blue. The kinase-binding motif responsible for both JAK2 and BRD4 affinity of TG10129 is shaded purple. (D) MLN8054 and the follow-on Aurora A candidate alisertib MLN8237; the benzodiazepine scaffold is highlighted in green; the structural differences in MLN8237 that abrogate GABA_A α-1 benzodiazepine binding are highlighted in red.

Figure 6

Probes That Elicit a Response in Biochemical or Cell-Based Assays Due to Non-Specific Effects (A) The metabolically unstable and chemically reactive components of iniparib and its reactive metabolite are depicted in red. (B) The substructures contributing to the lipophilicity of apoptazole are highlighted in lilac; the chemically reactive component of pifithrin-μ is highlighted in red. (C) Putative p53 modulator pifithrin-α, which undergoes rapid conversion to pifithrin-β. (D) The chemically reactive components of MCB-613 are highlighted in red. (E) Evolution of the early non-selective pathfinder tool LY294002 to the more potent and selective class I PI3 kinase inhibitor GDC-0941 (pictilisib).