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.

Autophagy is a conserved, lysosome-dependent process that maintains cellular homeostasis by degrading and recycling superfluous or damaged proteins and organelles. It involves the formation of double-membraned vesicles, termed autophagosomes, which trap cellular cargo and subsequently fuse with lysosomes, leading to degradation of their autophagic cargo. By limiting cellular damage and stress, basal autophagy exerts cytoprotective functions and prevents diverse pathologies, including cancer. However, the role of autophagy in cancer is highly complex and context-dependent. Certain types of tumor cells can hijack autophagy to sustain their high metabolic demands and to resist therapy-induced stress. Although preclinical studies have demonstrated that autophagy inhibition can sensitize tumors cells to conventional anticancer therapies, current clinical candidates, such as chloroquine and its derivatives, lack specificity and show limited efficacy. These limitations highlight the need for novel small-molecule autophagy inhibitors with improved potency, selectivity, and translational potential. To address this situation, we conducted a phenotypic high-throughput screening quantifying GFP-LC3-positive puncta to identify novel small-molecule autophagy modulators. Among the top-ranked hits, the biarylacetamide family emerged as promising candidates and was selected for further development. This thesis describes the optimization of the initial hits through three iterative rounds of structure-activity relationship. To explore the chemical space and improve potency we applied chemical modifications to biarylacetamide core in an explorative and diversity-oriented manner, generating a library of novel derivatives. The compounds were screened using the tandem fluorescent-tagged RFP-GFP-LC3 reporter in HeLa cells. The inhibitory activity of compounds that significantly increased RFP-GFP-LC3 puncta number was validated by immunoblot. These compounds led to an accumulation of the autophagic markers LC3, GABARAP and p62/SQSTM1 in both HeLa and LNCaP cells. Moreover, autophagic cargo flux assays, including LDH sequestration and LDHB-mKeima, further confirmed that these compounds inhibit autophagy at a post-sequestration step. To explore the biological effects induced by the biarylacetamides, we performed quantitative proteome profiling combined with untargeted metabolomic and lipidomic studies. These studies revealed that the biarylacetamides significantly alter lipid metabolism, including cholesterol biosynthesis pathway upregulation and changes in the distribution of key lipid classes. Further mechanistic studies indicated that the biarylacetamides triggered an ER stress response and may impair lysosomal function. Collectively these findings establish the biarylacetamides as a novel class of autophagy inhibitors. Ongoing target identification studies will define their mode of action and clarify how they disrupt autophagy. Evaluation of the biarylacetmides in combination with anticancer agents will help support their use as pharmacological tools and therapeutic candidates.