Structure-guided design of an LC3B-targeting PNA oligomer modulating autophagy and LC3B-mediated mRNA decay

: Autophagy plays a central role in cellular degradation and recycling pathways, and the LC3B protein is essential for this process. Dysregulation of LC3B has been implicated in oncogenesis. In this study, we used a computational approach to design a novel peptide nucleic acid (PNA) targeting the RNA-binding domain of LC3B, with the aim of inhibiting its function. The RNA AAUAAA polyadenylation signal was used as a starting point to design new PNAs with high affinity for LC3B. Molecular dynamics simulations and binding free-energy calculations on the RNA-AAUAAA/LC3B complex enabled the identification of promising PNA analogues. The lead candidate, a PNA with the sequence AATAAA, was synthesized, and its biological activity was investigated through biophysical and cellular assays. Cell viability was evaluated in the human prostate cell lines PNT1A and PNT2, as well as in the prostate cancer cell lines PC3 and DU145, while its efficacy in inhibiting autophagy was assessed in PC3 cells. The combined computational, biophysical and cellular results support AATAAA-PNA as an LC3B-binding hit whose cellular effects are consistent with modulation of LC3B-associated autophagy and mRNA-decay pathways. Overall, this study presents an in silico strategy for the design and development of LC3B inhibitors based on the RNA AAUAAA motif. The designed PNA represents a promising hit compound for further optimization in cancer therapy.