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Lysosomal protein degradation via autophagy strictly regulates cellular protein homoeostasis. Herein we performed high-content screening to identify compounds that inhibit autophagy pathways. We obtained 11 hit compounds and performed cluster analysis using cellular morphological information. Vacuolin-1, which induces the formation of giant vacuoles and is a target unknown compound, clustered with the known PIKfyve inhibitor YM201636. We further confirmed that vacuolin-1 is a potent PIKfyve inhibitor, and we finally concluded that PIKfyve inhibitors are novel chemical tools for regulating autophagy.

Elucidating the bioactive compound modes of action is crucial for increasing success rates in drug development. For anticancer drugs, defining effective drug combinations that overcome resistance improves therapeutic efficacy. Herein, by using a biologically annotated compound library, we performed a large-scale combination screening with Stearoyl-CoA desaturase-1 (SCD1) inhibitor, T-3764518, which partially inhibits colorectal cancer cell proliferation. T-3764518 induced phosphorylation and activation of AMPK in HCT-116 cells, which led to blockade of downstream fatty acid synthesis and acceleration of autophagy. Attenuation of fatty acid synthesis by small molecules suppressed the growth inhibitory effect of T-3764518. In contrast, combination of T-3764518 with autophagy flux inhibitors synergistically inhibited cellular proliferation. Experiments using SCD1 knock-out cells validated the results obtained with T-3764518. The results of our study indicated that activation of autophagy serves as a survival signal when SCD1 is inhibited in HCT-116 cells. Furthermore, these findings suggest that combining SCD1 inhibitor with autophagy inhibitors is a promising anticancer therapy.

BackgroundIn drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential.Results2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization.ConclusionThese results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0066-9) contains supplementary material, which is available to authorized users.