Computational Design of Miniproteins Targeting an NRAS-Derived pMHC Complex

The interaction between peptide-MHC complexes (pMHCs) and T cell receptors (TCRs) plays a key role in coordinating immune synapses. TCRs are capable of detecting various pMHCs, a characteristic known as cross-reactivity. While this allows the immune system to detect a wide range of threats, it can be risky in TCR-based therapies, since TCRs can inadvertently attack healthy cells, leading to serious side effects. When it comes to cancer-related mutations, such as those in the NRAS gene, these mutations produce new peptides (also known as neoantigens), that can be targeted by treatments given their similarity to self-peptides. In order to mitigate this type of concern in TCR-based therapies, it is essential to develop new strategies that are highly specific for neoantigens. Therefore, the objective of this study was to design and evaluate in silico specific miniproteins for the neoantigen NRAS Q61K presented by the MHC-I allele HLA*01:01. To this end, we used a pipeline that integrates PatchDock software for low-resolution molecular docking of 22 protein scaffolds (miniproteins) against the MHC-I allele HLA*01:01 complexed with the NRAS Q61K peptide (PDB 6MPP). After selecting the structures, we used RosettaScripts software to integrate high-resolution molecular docking cycles between the selected miniproteins and the pMHC complex followed by sequence sampling of the mini-binder residues at the interface, and energy minimization. The results demonstrated that the low-resolution molecular docking protocol was able to identify, among the 22 initial structures, miniproteins with optimized interaction profiles at the pMHC interface. These structures, in addition to being stable, also have diversified secondary structure profiles, which reinforces the possibility of interactions with the neoantigen. The interface design protocol, in turn, was able to redesign the interface residues of these miniproteins, optimizing the interactions with the pMHC. In conclusion, the designed binders formed more contacts and hydrogen bonds with critical peptide residues, reinforcing the specific recognition of the neoantigen, thus supporting the development of safer and more effective therapeutic strategies.