Abstract

Chaperone-Mediated Autophagy (CMA) is a selective lysosomal degradation process in which proteins bearing the KFERQ motif are recognized by the chaperone HSC70 and translocated across the lysosomal membrane via LAMP-2A, a transmembrane protein. This process is crucial for cellular homeostasis but is frequently upregulated in cancer cells, aiding their survival under stress conditions like hypoxia and nutrient deprivation. Inhibition of CMA has been linked to reduced tumor growth and metastatic potential, making it a promising target in cancer therapy. In this study, we aimed to develop novel inhibitors of the HSC70:LAMP-2A complex to block CMA selectively in cancer cells. Using comparative modeling and molecular dynamics simulations, we constructed models of HSC70 and LAMP-2A and predicted their interaction through protein-protein docking. The resulting complex was embedded in a lysosomal membrane model and subjected to molecular dynamics simulations in GROMACS, confirming the stability of the interaction site for inhibitor targeting. We screened over 3 million compounds from the MCULE and NCI databases for high binding affinity to the HSC70 interaction region. After filtering for ADME properties and structural diversity, several compounds were identified as potential inhibitors. Among these, three exhibited micromolar IC50 values in viability assays against several breast and colon cancer cell lines, while showing around 10-fold reduced toxicity in non-cancerous cells, demonstrating their potential as selective CMA inhibitors for cancer treatment. This work introduces CMA inhibition as a novel therapeutic approach, with specific compounds advancing as candidates for further analog development and preclinical evaluation.