De Novo Protein Design Enables Targeting of Intractable Oncogenic Protein-Protein Interfaces.

BACKGROUND/

OBJECTIVES: Protein-protein interactions (PPIs) involving oncogenic drivers remain among the most intractable targets in cancer biology due to their dynamic conformations and limited accessibility to conventional small molecules. Although antibodies and inhibitors have achieved clinical success against targets such as PD-1/PD-L1 and MYC, challenges persist related to tissue penetration, intracellular delivery, resistance, and incomplete blockade of key interface hotspots. The objective of this study is to develop an integrated computational framework for systematically designing hotspot-conditioned de novo miniprotein binders to target these interfaces.

METHODS: We present DesignForge, a computational protein design pipeline that integrates energetic hotspot identification, generative backbone design, sequence optimization, and structural confidence evaluation. The framework combines hotspot mapping using an open force-field-based energetic analysis module with generative backbone sampling using BindCraft, sequence optimization using ProteinMPNN, and structural validation using AlphaFold2. This in silico pipeline was applied to three representative oncogenic interfaces: PD-1/PD-L1, MYC/MAX, and KRAS/RAF.

RESULTS: Computationally generated designs exhibited high predicted structural confidence, favorable interface energetics, and consistent engagement of identified hotspot residues across targets. AlphaFold2-Multimer structural modeling indicated that the candidate PD-1 mimetic scaffolds, MYC/MAX interface binders, and KRAS interaction candidates can adopt conformations compatible with the target interfaces. Energetic contact analysis further supported predicted engagement of key hotspot residues. These findings support the computational feasibility of hotspot-conditioned binder generation using a unified design workflow.

CONCLUSIONS: DesignForge provides a reproducible computational framework for hotspot-guided de novo protein binder design targeting oncogenic protein-protein interfaces. The designs reported here represent computational predictions derived from structural modeling and energetic analysis. Experimental biochemical and cellular validation will be required to determine the functional activity of the proposed binders.