Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction

by Selleckchem | Jun 29 2023

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Comments:

The passage you provided describes a study that aimed to identify inhibitors of a specific protein-protein interaction (PPI) involved in the process of autophagy. Autophagy is a cellular process where cytosolic components are engulfed, degraded, and recycled. It has been implicated in cancer progression and inflammation.

The researchers focused on the PPI between two proteins, ATG12 and ATG3, which is crucial for autophagosome formation. They used a high throughput compound screening approach based on the protein-fragment complementation assay (PCA) to identify compounds that could inhibit this PPI. After screening over 41,000 compounds, they found 17 compounds that effectively inhibited the ATG12-ATG3 interaction in the PCA platform.

Next, they evaluated the ability of these compounds to inhibit autophagosome formation in viable cells. Among the tested compounds, they identified a lead compound labeled as #189. This compound showed inhibition of puncta formation of GFP-fused MAP1LC3B/LC3B, a protein associated with autophagy, with an IC50 value of 9.3 μM.

Further investigations revealed that compound 189 had a selective inhibitory effect on the growth of autophagy-dependent tumor cells. It also inhibited the secretion of IL1B/IL-1β, an inflammatory cytokine, by macrophage-like cells. These findings suggest that compound 189 may have therapeutic potential for cancer treatment and acute inflammatory responses.

The passage emphasizes the significance of targeting PPIs in autophagy for developing specific and selective inhibitors of autophagy. The abbreviations provided offer a quick reference for various terms used in the study.