Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design

Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs, or heterobifunctional degraders) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into close proximity. The resulting non-native protein-protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable POI-degrader-ligase ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the time-consuming and resource-intensive screening of the degrader linkers to induce favorable non-native PPIs between POI and E3 ligase. In this work, we present a physics-based computational protocol to systematically predict non-canonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the PROTAC ternary complex and enhance degradation. In our protocol, we build the non-Markovian dynamic model using the Integrative Generalized Master Equation (IGME) method from approximately 1.5 millisecond all-atom molecular dynamics (MD) simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue (KRAS) protein and the von Hippel-Lindau (VHL) E3 ligase. Our IGME model successfully revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included the thermodynamic and kinetic stabilities of these PPIs and the accessibility of the linker to the solvent-exposed sites on the warheads and the E3 ligand. One of our selected PPIs closely matches a recent co-crystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our IGME approach has significant potential for widespread application in predicting metastable POI-ligase encounter complex interfaces that can enable subsequent rational design of novel PROTACs.