Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supplementary ANIE-59-12470-s001.pdf (11M) GUID:?467A22B3-D293-4B93-B976-6B97F64C9CC4 Supplementary ANIE-59-12470-s002.avi (11M) GUID:?0840C9CA-626C-41A3-8AD0-354D22E02E2A Supplementary ANIE-59-12470-s003.avi (8.4M) GUID:?6286953B-731E-4F21-B81C-2BC5C70E24CA Abstract Pseudo\natural\product (NP) design combines STF 118804 natural product fragments to provide unprecedented NP\inspired compounds not accessible by biosynthesis, but endowed Rabbit Polyclonal to APBA3 with biological relevance. 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. alkenes was observed. Yields given over two steps. [a]?30?mol?% Pd(OAc)2, 72?h. [b]?10?mol?% Pd(OAc)2, 24?h. [c]?The iodoaniline HCl salt and 6.0?equiv. DABCO were used. [d]?Isolated as the TFA salt. =mean. All yields are unoptimized. A substructure search in the dictionary of natural products (DNP) revealed that both the indocinchona alkaloid scaffold and the fused quinuclidine\indole ring system were not found in NPs (see Figure?S1 in the Supporting Information for substructure searches). Comparison of the NP\likeness18 of the collection with the guiding Cinchona alkaloid NPs 1C4, NPs from ChEMBL,19 and drugs from DrugBank (Figure?2?c)20 revealed that the indocinchona alkaloids display a narrow NP\score distribution ([NP\likeness score]=?0.04), and contain connectivity that is more synthetic\compound\like than both the guiding Cinchona alkaloids (+0.83), and NPs in general (=+1.95). The average scores for the indocinchona alkaloids are close to the scores for compounds in DrugBank (=?0.01, and +0.02, respectively). Shape analysis of the compound collection by generating the two normalized principal moments of inertia values (Figure?2?d)21 indicated that the collection has a wide distribution of molecular shapes and a high three\dimensional character. The indocinchona alkaloids also exhibit a high fraction of sp3\hybridized carbon centers ([Fsp3]=0.30), a valuable feature for the successful progression of drug candidates.22 Overall the library has favorable properties for molecular discovery,23 with 96?% of the compounds falling within Lipinksi Rule\of\5 space (see Figure?S2).24 Since phenotypic screening enables identification of bioactivities of new small\molecule classes in an unbiased manner,26, 27, 28 we subjected STF 118804 the compound collection to a range of cell\based screens, including a phenotypic assay that monitors autophagic flux (see Table?1).29 Autophagy degrades and recycles both superfluous and damaged proteins and organelles by autophagosomes. Autophagy plays a crucial role in degenerative diseases and cancer, and novel small\ molecule autophagy inhibitors may provide inspiration for new drug discovery programs.30, 31 Table 1 Identification of Cinchona\alkaloid\inspired inhibitors of starvation and/or rapamycin\induced autophagy, derived from quinine.
1 10?h 5\CF3 7.860.8 n/a 2 10?i 5\NO2 7.331.5 n/a 3 10?k 5\OCF3 4.681.4 n/a 4 10?m 5\Cl 5.542.5 n/a 5 10?n 5\Br 6.781.1 n/a 6 10?r 6\CF3 5.823.0 n/a 7 10?s 6\Cl 8.121.5 n/a 8 10?u 7\OMe 2.460.6 2.370.7 9 10?w 7\azaindole 4.331.7 4.950.7 10 10?w\a 4\Cl\7\azaindole 0.520.20 0.650.35 11 10?w\b 5\Me\7\azaindole 0.310.09 0.860.26 12 10?w\c 5\Ar\7\azaindole 9.001.1 n/a 13 10?w\d 5\CF3\7\azaindole 0.120.03 0.770.29 14 10?w\e 5\NO2\7\azaindole 0.670.13 1.260.20 15 10?w\f 5\F\7\azaindole n/a n/a 16 10?w\g 5\Cl\7\azaindole 0.110.04 0.850.14 17 10?w\h 5\Br\7\azaindole 0.080.03 STF 118804 0.810.35 18 10?w\i 5\I\7\azaindole 0.080.02 1.240.20 19 10?w\j (azaquindole\1) 5\Br\6\Me\7\azaindole 0.040.02 0.100.02 20 10?w\k 6\Me\7\azaindole 3.120.5 5.111.4 21 10?w\l 6\Cl\7\azaindole 3.060.9 6.212.2 22 9?w 7\azaindole n/a nd 23 9?w\b 5\Me\7\azaindole n/a nd Open in a separate window All data are shown as mean SD of three independent experiments (N=3; n3). All compounds were initially assayed at a concentration of 10?m. For hits reducing the number of LC3 puncta by more than 50?%, IC50 values were determined. n/a=inactive (no reduction of LC3 puncta at 10?m). Ar=4\Cl\C6H4. nd=not determined. To identify autophagy inhibitors we monitored puncta formation in MCF7 cells stably transfected with an EGFP\tagged LC3 protein (MCF7\EGFP\LC3 cells) upon autophagy induction by amino\acid starvation, or treatment with the mTOR inhibitor rapamycin, using high\throughput automated image acquisition and analysis.32 Use of the autophagosome\autolysosome fusion inhibitor chloroquine (CQ) enhanced the dynamic range of the assay. The compounds 10, derived from quinine, inhibited starvation\induced autophagy (Table?1, entries?1C9), but no inhibitory activity was observed for the quinidine\derived indoles 9?aCw at 10?m. The compounds substituted at the 5\ and 6\positions of the indole ring.