Protein expression and function in eukaryotic cells are tightly harmonized processes modulated by the combination of different layers of regulation, including transcription, processing, stability, and translation of messenger RNA, as well as assembly, maturation, sorting, recycling, and degradation of polypeptides. The integration between all these pathways and the protein quality control machinery, deputed to avoid the production and accumulation of aberrantly folded proteins, determines protein homeostasis. Recent computational advancements in the simulation of biochemical processes open the possibility of investigating biological mechanisms with physics-based models. One of these methods, called Bias Functional, allows the reconstruction of protein folding and misfolding pathways at an atomistic level of resolution.1,2 By coupling this innovative computational technology with rigorous experimental techniques, we discovered the existence of functional, nonnative metastable states (folding intermediates) transiently appearing along the folding process of several proteins. We collected evidence indicating that protein folding intermediates could play a role in disparate biological processes, including post-translational regulation3 and host-pathogen interactions. Inspired by such an unexpected biological paradigm, we designed a novel drug discovery approach to selectively suppress target proteins by impairing their folding process rather than targeting their native conformations (named Pharmacological Protein Inactivation by Folding Intermediate Targeting, PPI-FIT).4,5 PPI-FIT was employed for the first time to identify a pharmacological degrader of the cellular prion protein (PrP), a cell surface glycoprotein playing a central role in fatal and transmissible neurodegenerative pathologies known as prion diseases.5,6 Our data reveal a previously unappreciated role for folding intermediates in the regulation of protein homeostasis and directly support the concept of modulating the expression of virtually any protein by acting on folding pathways.

References 1. Beccara, S. A., Fant, L. & Faccioli, P. Variational scheme to compute protein reaction pathways using atomistic force fields with explicit solvent. Phys. Rev. Lett. 114, 098103 (2015). 2. Spagnolli, G. et al. Full atomistic model of prion structure and conversion. PLoS Pathog. 15, e1007864 (2019). 3. Spagnolli, G., Massignan, T., Astolfi, A. et al. Pharmacological protein inactivation by targeting folding intermediates. BioRxiv doi: https://doi.org/10.1101/2020.03.31.018069 4. Spagnolli, G., Massignan, T., Astolfi, A. et al. Pharmacological inactivation of the prion protein by targeting a folding intermediate. Commun Biol 4, 62 2021). 5. Faccioli, P. and Biasini, E. Patent n. PCT/IB2019/056371, A method for identifying folding intermediates of proteins. 6. Biasini, E. Barreca, ML. Faccioli, P. and Mancini, I. Patent n. WO/2021/191883, Small molecules inducing the degradation of the cellular prion protein.