Razumevanje vloge strukturnih lastnosti ternarnih kompleksov pri ciljni razgradnji proteinov: Mehanistična študija

Oddelek UP FAMNIT, v okviru katerega se izvaja projekt:

Oddelek za aplikativno naravoslovje

The ever-growing need for improved therapies against cancer and other severe diseases calls for innovative approaches that move beyond the limitations of small-molecule inhibitors. Targeted protein degradation (TPD) offers a transformative solution by capitalizing on the cell’s ubiquitin–proteasome system to eliminate harmful or dysregulated proteins, rather than merely inhibiting them. Proteolysis-targeting chimeras (PROTACs)have emerged as a particularly promising TPD platform, simultaneously recruiting a protein of interest (POI) and an E3 ligase into a ternary complex that facilitates POI ubiquitination and subsequent proteasomal degradation. This mechanism has the potential to broaden the druggable proteome, address acquired drug resistance, and reduce off-target effects—key advantages over traditional inhibition strategies.

Yet despite the rapid progress and the increasing number of PROTACs in clinical trials, fundamental questions about how structural and conformational features of the fully assembled ternary complex drive selective and efficient ubiquitination still remain. Most current models rely on static crystal structures or simplified docking, rarely capturing the complete catalytic complex—including the loaded Ub~E2 enzyme—or the dynamic ensemble of states it can adopt. Consequently, many mechanistic nuances underlying substrate lysine selection, reaction specificity, and overall degradation potency remain elusive.

To address these gaps, our project integrates a synergistic suite of computational and experimental methods. We will employ state-of-the-art docking protocols and enhanced sampling molecular dynamics simulations to characterize the conformational landscape of the ternary complex and identify configurations responsible for productive ubiquitin transfer. We will also conduct hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to elucidate the atomistic pathways of the initial POI ubiquitination step (“priming”) in which specific POI lysine residues form iso-peptide bonds with ubiquitin. Complementary ubiquitinomics experiments will complement the computational predictions, pinpointing which lysine residues are ubiquitinated in vitro.

As a case study, we will investigate a series of SMARCA2/4 degraders with varying potencies and selectivities. This will enable us to uncover the structural factors—from linker length and geometry to binding-site interactions—that govern differential ubiquitination patterns, ultimately shedding light on how to rationally tune PROTACs for efficient POI targeting. By understanding the interplay between ternary complex structure, conformational flexibility, and catalytic efficiency, we aim to establish robust structure–activity relationships that will guide the next generation of TPD therapeutics.

In the course of this work, we will also develop an automated computational pipeline that integrates docking, advanced molecular simulations, and QM/MM modeling with high-throughput data analysis. Released as an open-source platform, this pipeline will serve as a valuable community resource for researchers seeking to design and optimize PROTACs against diverse protein targets. The combination of thorough mechanistic in sights and accessible computational tools is expected to accelerate TPD-focused drug discovery, ultimately advancing new avenues for treating diseases previously considered “undruggable.” We will disseminate our findings through open-access publications, presentations at scientific conferences, and outreach to the broader public, fostering collaboration across disciplines and ensuring maximal impact on the emerging field of TPD.