To determine the mode of VPS34 inhibition we investigated the kinetics for inhibition of VPS34 by azaquindole\1 (10?w\j), which indicated that it was an ATP competitive inhibitor (Number?4?c). Open in a separate window Figure 4 a)?Cellular thermal shift assay (CETSA) for the binding of azaquindole\1 (10?w\j) to VPS34 in cell lysate (green collection=VEH; red collection=10?w\j). exposed a novel class of azaindole\comprising autophagy inhibitors, the azaquindoles. Subsequent characterization of the most potent compound, azaquindole\1, in the morphological cell painting assay, guided target identification attempts. In contrast to the parent Cinchona alkaloids, azaquindoles selectively inhibit starvation\ and rapamycin\induced autophagy by focusing on the lipid kinase VPS34. alkenes was observed. Yields given over two methods. [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) exposed that both the indocinchona alkaloid scaffold and the fused quinuclidine\indole ring system were not found in NPs (observe Number?S1 in the Supporting Info for substructure searches). Comparison of the NP\likeness18 of the YUKA1 collection with the guiding Cinchona alkaloid NPs 1C4, NPs from ChEMBL,19 and medicines from DrugBank (Number?2?c)20 revealed the indocinchona alkaloids display a thin 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 ideals (Number?2?d)21 indicated the collection has a wide distribution of molecular designs and a high three\dimensional character. The indocinchona alkaloids also show a high portion 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 finding,23 with 96?% of the compounds falling within Lipinksi Rule\of\5 space (observe Number?S2).24 Since phenotypic screening enables recognition of bioactivities of new small\molecule classes in an unbiased manner,26, 27, 28 we subjected 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 takes on a crucial part in degenerative diseases and malignancy, and novel small\ molecule autophagy inhibitors may provide inspiration for new drug discovery programs.30, 31 Table 1 Recognition 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 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 windows All data are shown as mean SD of three independent experiments (N=3; n3). All compounds were assayed at a focus of 10 initially?m. For strikes lowering the real amount of LC3 puncta by a lot more than 50?%, IC50 beliefs were motivated. n/a=inactive (no reduced amount of LC3 puncta at 10?m). Ar=4\Cl\C6H4. nd=not really determined. To recognize autophagy inhibitors we supervised puncta development in MCF7 cells stably transfected with an EGFP\tagged LC3 proteins (MCF7\EGFP\LC3 cells) upon autophagy induction by amino\acidity hunger, or treatment using the mTOR inhibitor rapamycin, using high\throughput automated picture evaluation and acquisition.32 Usage of the autophagosome\autolysosome fusion inhibitor chloroquine (CQ) improved the dynamic selection of the assay. The substances 10, produced from quinine, inhibited hunger\induced autophagy (Desk?1, entries?1C9), but no inhibitory activity was observed for the quinidine\derived indoles 9?aCw in 10?m. The substances substituted on the 5\ and 6\positions from the indole band (entries?1C7) were relatively weak inhibitors of hunger\induced.The inhibition of autophagy had not been a task shared by simple indoles, 7\azaindoles, quinuclidines, or quinolines (see Figure?S5). kinase VPS34. alkenes was noticed. Yields provided over two guidelines. [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 had been utilized. [d]?Isolated as the TFA salt. =mean. All produces are unoptimized. A substructure search in the dictionary of natural basic products (DNP) uncovered that both indocinchona alkaloid scaffold as well as the fused quinuclidine\indole band system weren’t within NPs (discover Body?S1 in the Helping Details for substructure queries). Comparison from the NP\likeness18 from the collection using the guiding Cinchona alkaloid NPs 1C4, NPs from ChEMBL,19 and medications from DrugBank (Body?2?c)20 revealed the fact that indocinchona alkaloids screen a slim NP\rating distribution ([NP\likeness rating]=?0.04), and contain connection that’s more man made\substance\like than both guiding Cinchona alkaloids (+0.83), and NPs generally (=+1.95). The common ratings for the indocinchona alkaloids are near to the ratings for substances in DrugBank (=?0.01, and +0.02, respectively). Form analysis from the substance collection by producing both normalized principal occasions of inertia beliefs (Body?2?d)21 indicated the fact that collection includes a wide distribution of molecular styles and a higher three\dimensional personality. The indocinchona alkaloids also display a high small fraction of sp3\hybridized carbon centers ([Fsp3]=0.30), a very important feature for the successful development of drug applicants.22 Overall the collection has favorable properties for molecular breakthrough,23 with 96?% from the substances dropping within Lipinksi Guideline\of\5 space (discover Body?S2).24 Since phenotypic testing enables id of bioactivities of new little\molecule classes within an unbiased way,26, 27, 28 we subjected the compound collection to a variety of cell\based displays, including a phenotypic assay that monitors autophagic flux (see Desk?1).29 Autophagy degrades and recycles both superfluous and broken proteins and organelles by autophagosomes. Autophagy has a crucial function in degenerative illnesses and tumor, and novel little\ molecule autophagy inhibitors might provide motivation for new medication discovery applications.30, 31 Desk 1 Id of Cinchona\alkaloid\inspired inhibitors of starvation and/or rapamycin\induced autophagy, produced from quinine.
1 10?h 5\CF3 7.860.8 n/a 2 10?we 5\Zero2 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 personally\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\Zero2\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 0.810.35 18 10?w\we 5\We\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 personally\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 personally\7\azaindole n/a nd Open up in another home window All data are shown as mean SD of 3 independent tests (N=3; n3). All substances were primarily assayed at a focus of 10?m. For strikes reducing the amount of LC3 puncta by a lot more than 50?%, IC50 beliefs were motivated. n/a=inactive (no reduced amount of LC3 puncta at 10?m). Ar=4\Cl\C6H4. nd=not really determined. To recognize autophagy inhibitors we supervised puncta development in MCF7 cells stably transfected with an EGFP\tagged LC3 proteins (MCF7\EGFP\LC3 cells) upon autophagy induction by amino\acidity hunger, or treatment using the mTOR inhibitor rapamycin, using high\throughput computerized picture acquisition and evaluation.32 Usage of the autophagosome\autolysosome fusion inhibitor chloroquine (CQ) improved the.Notably, autophinib can be a known VPS34 inhibitor, as well as the cell painting analysis validates this previously finding. painting assay, led target identification attempts. As opposed to the mother or father Cinchona alkaloids, azaquindoles selectively inhibit hunger\ and rapamycin\induced autophagy by focusing on the lipid kinase VPS34. alkenes was noticed. Yields provided over two measures. [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 had been utilized. [d]?Isolated as the TFA salt. =mean. All produces are unoptimized. A substructure search in the dictionary of natural basic products (DNP) exposed that both indocinchona alkaloid scaffold as well as the fused quinuclidine\indole band system weren’t within NPs (discover Shape?S1 in the Helping Info for substructure queries). Comparison from the NP\likeness18 from the collection using the guiding Cinchona alkaloid NPs 1C4, NPs from ChEMBL,19 and medicines from DrugBank (Shape?2?c)20 revealed how the indocinchona alkaloids screen a slim NP\rating distribution ([NP\likeness rating]=?0.04), and contain connection that’s more man made\substance\like than both guiding Cinchona alkaloids (+0.83), and NPs generally (=+1.95). The common ratings for the indocinchona alkaloids are near to the ratings for substances in DrugBank (=?0.01, and +0.02, respectively). Form analysis from the substance collection by producing both normalized principal occasions of inertia ideals (Shape?2?d)21 indicated how the collection includes a wide distribution of molecular styles and a higher three\dimensional personality. The indocinchona alkaloids also show a high small fraction of sp3\hybridized carbon centers ([Fsp3]=0.30), a very important feature for the successful development of drug applicants.22 Overall the collection has favorable properties for molecular finding,23 with 96?% from the substances dropping within Lipinksi Guideline\of\5 space (discover Shape?S2).24 Since phenotypic testing enables recognition of bioactivities of new little\molecule classes within an unbiased way,26, 27, 28 we subjected the compound collection to a variety of cell\based displays, including a phenotypic assay that monitors autophagic flux (see Desk?1).29 Autophagy degrades and recycles both superfluous and broken proteins and organelles by autophagosomes. Autophagy takes on a crucial part in degenerative illnesses and tumor, and novel little\ molecule autophagy inhibitors might provide motivation for new medication discovery applications.30, 31 Desk 1 Recognition of Cinchona\alkaloid\inspired inhibitors of starvation and/or rapamycin\induced autophagy, produced from quinine.
1 10?h 5\CF3 7.860.8 n/a 2 10?we 5\Zero2 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 personally\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\Zero2\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 0.810.35 18 10?w\we 5\We\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 personally\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 Rabbit Polyclonal to MINPP1 9?w\b 5\Me personally\7\azaindole n/a nd Open up in another screen All data are shown as mean SD of 3 independent tests (N=3; n3). All substances were originally assayed at a focus of 10?m. For strikes reducing the amount of LC3 puncta by a lot more than 50?%, IC50 YUKA1 beliefs were driven. n/a=inactive (no reduced amount of LC3 puncta at 10?m). Ar=4\Cl\C6H4. nd=not really determined. To recognize autophagy inhibitors we supervised puncta development in MCF7 cells stably transfected with an EGFP\tagged LC3 proteins (MCF7\EGFP\LC3 cells) upon autophagy induction by amino\acidity hunger, or treatment using the mTOR inhibitor rapamycin, using high\throughput computerized picture acquisition and evaluation.32 Usage of the autophagosome\autolysosome fusion inhibitor chloroquine (CQ) improved the dynamic selection of the assay. The substances 10, produced from quinine, inhibited hunger\induced autophagy (Desk?1, entries?1C9), but no inhibitory activity was observed for the quinidine\derived indoles 9?aCw in 10?m. The substances substituted on the 5\ and 6\positions from the indole band (entries?1C7) were relatively YUKA1 weak inhibitors of hunger\induced autophagy (IC504.7C8.1?m), and the ones substituted with polar functionalities on the 7\position from the indole band (entries?8 and 9) gave appreciably higher actions. Hence, the 7\azaindole\substituted substance 10?w inhibited hunger\induced autophagy with IC50=4.331.7?m, whilst the 7\methoxy\substituted indole 10?u gave one of the most dynamic initial substance with IC50=2.460.6?m. Notably, the last mentioned two substances (entries?8 and 9) also inhibited rapamycin\induced autophagy, recommending that they respond either downstream or of mTOR independently. Generally, 7\azaindoles had been potent, sub\micromolar inhibitors of autophagy (entries?10C21. Find Desk?S1 for.We thank Oliver Bartels for regimen chemical substance synthesis. inhibitors, the azaquindoles. Following characterization of the very most potent substance, azaquindole\1, in the morphological cell painting assay, led target identification initiatives. As opposed to the mother or father Cinchona alkaloids, azaquindoles selectively inhibit hunger\ and rapamycin\induced autophagy by concentrating on the lipid kinase VPS34. alkenes was noticed. Yields provided over two techniques. [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 had been utilized. [d]?Isolated as the TFA salt. =mean. All produces are unoptimized. A substructure search in the dictionary of natural basic products (DNP) uncovered that both indocinchona alkaloid scaffold as well as the fused quinuclidine\indole band system weren’t within NPs (find Amount?S1 in the Helping Details for substructure queries). Comparison from the NP\likeness18 from the collection using the guiding Cinchona alkaloid NPs 1C4, NPs from ChEMBL,19 and medications from DrugBank (Amount?2?c)20 revealed which the indocinchona alkaloids screen a small NP\rating distribution ([NP\likeness rating]=?0.04), and contain connection that’s more man made\substance\like than both guiding Cinchona alkaloids (+0.83), and NPs generally (=+1.95). The common ratings for the indocinchona alkaloids are near to the ratings for substances in DrugBank (=?0.01, and +0.02, respectively). Form analysis from the substance collection by producing both normalized principal occasions of inertia beliefs (Amount?2?d)21 indicated which the collection includes a wide distribution of molecular forms and a higher three\dimensional personality. The indocinchona alkaloids also display a high small percentage of sp3\hybridized carbon centers ([Fsp3]=0.30), a very important feature for the successful development of drug applicants.22 Overall the collection has favorable properties for molecular breakthrough,23 with 96?% from the substances dropping within Lipinksi Guideline\of\5 space (find Amount?S2).24 Since phenotypic testing enables id of bioactivities of new little\molecule classes within an unbiased way,26, 27, 28 we subjected the compound collection to a variety of cell\based displays, including a phenotypic assay that monitors autophagic flux (see Desk?1).29 Autophagy degrades and recycles both superfluous and broken proteins and organelles by autophagosomes. Autophagy has a crucial function in degenerative illnesses and cancers, and novel little\ molecule autophagy inhibitors might provide motivation for new medication discovery applications.30, 31 Desk 1 Id of Cinchona\alkaloid\inspired inhibitors of starvation and/or rapamycin\induced autophagy, produced from quinine.
1 10?h 5\CF3 7.860.8 n/a 2 10?we 5\Zero2 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 personally\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\Zero2\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 0.810.35 18 10?w\we 5\We\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 personally\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 personally\7\azaindole n/a nd Open in a separate windows All data are shown as mean SD of three independent experiments (N=3; n3). All compounds were in the beginning assayed at a concentration of 10?m. For hits reducing the number of LC3 puncta by more than 50?%, IC50 values were decided. 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\.Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supplementary Click here for additional data file.(11M, pdf) Supplementary Click here for additional data file.(11M, avi) Supplementary Click here for additional data file.(8.4M, avi) Acknowledgements D.J.F. by targeting the lipid kinase VPS34. alkenes was observed. Yields given over two actions. [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 (observe Physique?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 (Physique?2?c)20 revealed that this indocinchona alkaloids display a thin 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 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 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.