Synthesis of a 10,000-membered library of molecules resembling carpanone and discovery of vesicular traffic inhibitors

Abstract

Split-and-pool synthesis of a 10,000-membered library of molecules resembling the natural product carpanone has been achieved. The synthesis features development of solid-phase multicomponent reactions between nitrogen nucleophiles, enones, and hydroxylamines, and a solid-phase application of the Huisgen cycloaddition affording substituted triazoles. The synthesis was performed in high-capacity (500 microm) polystyrene beads using a one bead-one stock solution strategy that enabled phenotypic screens of the resulting library. Using whole-cell fluorescence imaging, we discovered a series of molecules from the carpanone-based library that inhibit exocytosis from the Golgi apparatus. The most potent member of this series has an IC(50) of 14 microM. We also report structure-activity relationships for the molecules exhibiting this interesting phenotype. These inhibitors of exocytosis may be useful reagents for the study of vesicular traffic.

Figure 1

High-throughput screening of a library of molecules resembling carpanone for inhibitors of VSVG^ts-GFP traffic. (A) Statistics of the hits that scored in the original screen: nine compounds were identified that block VSVG^ts-GFP exit from the Golgi, and four compounds were identified that cause Golgi fragmentation. Also, many compounds exhibited nonspecific phenotypes that were mixtures of ER block and Golgi block. (B) Schematic illustrating the phenotype for the class of compounds that inhibits VSVG^ts-GFP exit from the Golgi. (C) Representative examples of the hits from the original screen and their derivatives that were resynthesized. (D) Representative dose response profiles for two of the most potent hits from the library. Also shown is the inactive carpanone natural product. (E) Structure-activity relationships (SAR) for the various hits at R³. Variations in this series did not significantly perturb the potency of the compounds, with the hydroxy and allyloxy groups showing the highest potencies compared to that of the o-fluorobenzyloxy group. (F) SAR of hits varying at position R². In this series, potency decreases when the secondary amine is replaced with an ethyl carbamate or a benzyloxy ethyl carbamate. The methyl oxime was least potent regardless of the identity of the R² group.

Figure 2

Hit CLL-19, identified from a screen of a library of molecules resembling carpanone, is a specific inhibitor of VSVG^ts–GFP traffic. (A) Endocytosis experiments illustrating that CLL-19 (33 μM) does not have an effect on the uptake of fluorescently labeled cholera toxin (CT-594) or transferrin. Also shown are immunofluorescence experiments demonstrating that CLL-19 has no effect on the steady-state dynamics of actin or tubulin. (B) Immunofluorescence experiments probing the effect of CLL-19 on various cellular organelles. Three-hour treatment of 33 μM CLL-19 does not affect the intracellular distribution of the ER exit sites marker sec13p, and the cis-Golgi marker GM130, suggesting that the architectural integrity of the Golgi is maintained. The KDEL receptor, which recycles between the ER and the Golgi, appears slightly condensed on treatment with CLL-19 compared to the control, while clathrin distribution appears normal.

Figure 3

Effect of CLL-19 on cellular division and cell cycle progression. (A) Mitotic index experiments measuring the percent of cells in mitosis after 6 h treatment with 33 μM CLL-19 or 33 μM nocodazole. (B) Flow cytometry experiments showing the decrease in the percent of cells in the G2/M phase and their increase in the S phase following 6 h treatment with CLL-19. Nocodazole is shown as a control.

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Chart 1

Natural Products and Their Derivatives Used as Medicines or to Probe Biology

Chart 2

Compilation of Building Blocks Used in the Library Synthesis