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.

Design, Synthesis, and Biological Evaluation of Chemically and Biologically Diverse Pyrroquinoline Pseudo Natural Products

“…For the visual comparison of the data, fingerprints were assembled and the induction value (fraction of parameters that underwent significant changes compared to the DMSO control; see the SI for further information) was computed. Fingerprints have previously been used for the identification of possible mechanisms of action through comparison to compounds with annotated targets and activity [16–18] . We used these fingerprints and their underlying data as inputs for principal component analysis (PCA) to gauge the similarity of bioactivities within and between compound classes.…”

“…Fingerprints have previously been used for the identification of possible mechanisms of action through comparison to compounds with annotated targets and activity. [16][17][18] We used these fingerprints and their underlying data as inputs for principal component analysis (PCA)togauge the similarity of bioactivities within and between compound classes.T his approach has previously been used by Schreiber et al to demonstrate differences in cellular responses to stereoisomers [36] and Aldrich et al to illustrate the diverse performance of flavonoid-inspired libraries. [37,38] In general, the different scaffolds induced pronounced morphological changes,w hich is expressed in the induction value.Most scaffolds displayed low induction values (< 10 %, Table S3) at 10 mMc ompound concentration except for the unsaturated PQs H.Members of this compound class showed strong changes in cell morphology and induction values up to 62 %.…”

“…The library synthesis includes the discovery of a new methodology for oxidative cycloadditions to quinolinium salts. Cheminformatic analysis and biological evaluation of the compound collection by means of phenotyping in the morphological “cell painting” assay [11–19] revealed that the pseudo‐NP sub‐classes are chemically and biologically diverse. The NP‐likeness [20] and bioactivity patterns differ markedly between the eight scaffolds, and depend on connectivity and regioisomeric arrangement of the fragments in the isomeric pseudo‐NP classes.…”

“…Here we provide proof-of-principle for this notion by means of design, synthesis,c heminformatic characterization and biological evaluation of a155-membered pyrroquinoline (PQ) pseudo-NP collection which combines fragments characteristic for the biosynthetically unrelated tetrahydroquinoline-and pyrrolidine NP classes in eight different connectivities and regioisomeric arrangements.T he library synthesis includes the discovery of an ew methodology for oxidative cycloadditions to quinolinium salts.C heminformatic analysis and biological evaluation of the compound collection by means of phenotyping in the morphological "cell painting" assay [11][12][13][14][15][16][17][18][19] revealed that the pseudo-NP sub-classes are chemically and biologically diverse.T he NP-likeness [20] and bioactivity patterns differ markedly between the eight scaffolds, and depend on connectivity and regioisomeric arrangement of the fragments in the isomeric pseudo-NP classes.…”

Identifying bioactivity of pseudo-natural products using the Cell Painting assay

“…Another example where Cell Painting supported the target elucidation process was reported by Foley et al 40 In their pseudo-NPs the cinchona alkaloid scaffold is combined with indoles and aza-indoles, forming previously undescribed indocinchona alkaloids (Figure 5).…”