Fully functionalized small-molecule probes for integrated phenotypic screening and target identification

Abstract

Phenotypic screening offers a powerful approach to identify small molecules that perturb complex biological processes in cells and organisms. The tendency of small molecules, however, to interact with multiple protein targets, often with moderate to weak affinities, along with the lack of straightforward technologies to characterize these interactions in living systems, has hindered efforts to understand the mechanistic basis for pharmacological activity. Here we address this challenge by creating a fully functionalized small-molecule library whose membership is endowed with: (1) one or more diversity elements to promote interactions with different protein targets in cells, (2) a photoreactive group for UV light-induced covalent cross-linking to interacting proteins, and (3) an alkyne handle for reporter tag conjugation to visualize and identify cross-linked proteins. A library member was found to inhibit cancer cell proliferation selectively under nutrient-limiting (low glucose) conditions. Quantitative chemoproteomics identified MT-ND1, an integral membrane subunit of the ∼1 MDa NADH:ubiquinone oxidoreductase (complex 1) involved in oxidative phosphorylation, as a specific target of the active probe. We further demonstrated that the active probe inhibits complex 1 activity in vitro (IC(50) = 720 nM), an effect that is known to induce cell death in low-glucose conditions. Based on this proof of principle study, we anticipate that the generation and integration of fully functionalized compound libraries into phenotypic screening programs should facilitate the discovery of bioactive probes that are amenable to accelerated target identification and mechanistic characterization using advanced chemoproteomic technologies.

Figure 1

A fully functionalized small-molecule library for integrated cell-based screening and target identification. (A) BzIndole and BzBd scaffolds and structures of repre-sentative library members. See Figure S1 for structures of all synthesized members of the library. (B) Proteomic profiles for library members (10 μM) following in situ photocrosslinking in MDA-MB-231 cells. Probe-labeled proteins were visualized by click chemistry conjugation to an azide-rhodamine tag, SDS-PAGE, and in-gel fluorescence scanning (fluorescent gels shown in gray scale).

Figure 2

Anti-proliferative activity of compound 1 in human cancer cell lines. (A) Inhibition of cancer cell viability in low glucose (1 mM) media after 1 treatment (24 hr) as determined by the WST-1 assay. (B) Comparison of cell viability in low (1 mM) and normal (8 mM) glucose for cells treated with 10 μM active (1) or control (14) probes, or vehicle (DMSO). Each cell line’s viability was normalized to DMSO-treated cells grown in 8 mM glucose. See Figure S3 for complete screening results.

Figure 3

Chemoproteomic identification of proteins that interact with probe 1 in cancer cells. (A) A nonclickable analog of 1 (compound 37) for competition profiling experiments (reagents: 1 atm H₂, cat. Pd–BaSO₄, EtOH). (B) Gel profile comparing proteins labeled by 1 and 14 (10 μM) and 1 competed with 5X 37 (50 μM). Candidate protein bands corresponding to selective targets of 1 identified by LC-MS are denoted on right. (C) Extracted MS1 chromatographs for a representative peptide (ILGYMQLR, 497.28114 m/z) from MT-ND1 shown for 1 +/− 5X 37 competition, 1 versus 1, 1 versus 14, and 1 versus DMSO (unlabeled) experiments. See Table 1, Figure S5, and Tables S1 and S2 for more complete presentations of the quantitative proteomic data. (D) Assay for complex 1 NADH oxidase showing selective inhibition by 1 over 14.