Figure 1.

Identification of molecular targets by photo-affinity labeling and click chemistry. (A) Overview of photo-labile click probe (PLCP) design, photo-affinity labeling and click bioconjugation using a fluorescent reporter probe. (B) Cell-based strategy for click-based molecular target identification for trans-translation inhibitors. The process involves intracellular probe-target binding, intracellular probe-target covalent cross-linking, cell lysis, click bioconjugation using either a secondary fluorescent click probe (SFCP) or secondary affinity click probe (SACP), target isolation, identification by peptide mass fingerprinting (PMF), and validation all leading to lead optimization. Abbreviations: AAHC, Azide-alkyne Huisgen Cycloaddition; FL, fluorescence-exposed gel; Gel lanes, one ladder; two cell lysate; three target isolated from a click-based affinity pull-down assay; 4 target isolated from a click-based fluorescence assay.