Phenotyping Reveals Targets of a Pseudo‐Natural‐Product Autophagy Inhibitor

Abstract

Pseudo‐natural‐product (NP) design combines natural product fragments to provide unprecedented NP‐inspired compounds not accessible by biosynthesis, but endowed with biological relevance. Since the bioactivity of pseudo‐NPs may be unprecedented or unexpected, they are best evaluated in target agnostic cell‐based assays monitoring entire cellular programs or complex phenotypes. Here, the Cinchona alkaloid scaffold was merged with the indole ring system to synthesize indocinchona alkaloids by Pd‐catalyzed annulation. Exploration of indocinchona alkaloid bioactivities in phenotypic assays revealed a novel class of azaindole‐containing autophagy inhibitors, the azaquindoles. Subsequent characterization of the most potent compound, azaquindole‐1, in the morphological cell painting assay, guided target identification efforts. In contrast to the parent Cinchona alkaloids, azaquindoles selectively inhibit starvation‐ and rapamycin‐induced autophagy by targeting the lipid kinase VPS34.

Natural product fragment combination to performance-diverse pseudo-natural products

“…The quinine- or quinidine-derived ketone (1.0 equiv), a 2-iodoaniline derivative (3.0 equiv), DABCO (3.0 equiv) and Pd(OAc) 2 (0.1 equiv) were added to a reaction vial equipped with a screw-cap lid 6 . The reactants were dissolved in anhydrous DMF (1.0 ml / 0.32 mmol) and degassed under a stream of Argon for 10 min then heated to 105 °C.…”

“…The design and synthesis of pseudo-natural products (PNPs) through the combination of natural product (NP) fragments is a recently proposed principle for the discovery of bioactive compounds 1 – 6 . The concept aims to synthetically combine fragments of NPs that are biosynthetically unrelated and have different bioactivity to produce scaffolds that resemble NPs but are not accessible through existing biosynthetic pathways.…”

“…Comparison to reference compounds with known bioactivities can give insight into the mode of action and/or biological target(s). Cell painting has been employed to differentiate bioactivity profiles based on structural variances and chemical properties 12 , 13 , to identify small molecule targets 6 , to identify mode of action even when the target is not a protein 14 , and to delineate qualitative structure-activity relationships 4 .…”

Reference compounds for characterizing cellular injury in high-content cellular morphology assays

“…The CP assay has been used for its strong information content while being higher in throughput and lower in cost relative to other profiling techniques like transcriptomics 9 . Mechanistic hypotheses can be inferred from CP data when compounds share similar phenotypic profiles [10][11][12][13] . Many groups have used CP to biologically annotate novel synthetic and other chemical libraries [14][15][16][17][18] .…”

Virtual screening for small molecule pathway regulators by image profile matching

“…Evidence in favor of the strategy include anecdotal cases where perturbations of genes phenocopy the compounds targeting them 10 , though these usually were done in the context of having a compound of interest and looking for the target instead of vice versa. Almost two decades of research indicates that image profiles can identify compounds inducing phenotypes that match other compounds 11 (reviewed in 9 ); this can be helpful in determining a compound's mechanism of action (if the matching compounds are annotated) [12][13][14][15][16] and in identifying novel chemical structures with behavior similar to known compounds with desired bioactivity. This latter case can be useful for finding compounds with better physical-chemical or physiological properties, or working around intellectual property concerns, but it requires having a compound with the desired biological impact already.…”