Targeting autophagy is believed to hold great promise for the treatment of various diseases, including cancer. However, since the therapeutic efficacy of currently available autophagy-modulating drugs is limited by off-target effects and the requirement of high dosage, there is an urgent need to develop novel autophagy-targeting compounds. In this study, we report molecules of the biarylacetamide class as novel autophagy inhibitors. These molecules were identified via phenotypic high-throughput screening, and a series of analogues was subsequently synthesized. Among these, 5d and 5j were retained as potent autophagy blockers in HeLa and LNCaP cells. Both compounds inhibited autophagy at a late-stage in the pathway, as evidenced by the strong accumulation of RFP-GFP-LC3 puncta as well as LC3-II, GABARAP-II and SQSTM1 protein levels, resembling the effects obtained with the well-known late-stage autophagy inhibitor Bafilomycin A1. Quantitative proteome profiling combined with metabolomic and lipidomic studies revealed that 5j significantly altered lipid metabolism. These alterations included activation of the cholesterol biosynthesis pathway and changes in the distribution of key lipid classes, such as phospholipids, ceramides and triglycerides. Further mechanistic studies indicated that 5d and 5j triggered an ER stress response and may impair lysosomal function, as suggested by the accumulation of pro-cathepsin D. Collectively, these findings demonstrate that 5j is a novel and potent late-stage autophagy inhibitor with a distinct mechanism of action compared to currently available inhibitors.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a ‘hit’ during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.