The KRAS oncoprotein remains a therapeutic challenge due to the limitations
of current covalent inhibitors. This work presents the rational design of PIAKRASv2-
Nb, a 100 % humanized nanobody generated via the Protein Interaction
Architect (PIA) method, which binds to the DEYDPTIEDS epitope in the
Switch I region of KRAS with a high predicted affinity (ipTM = 0.78). Unlike
classical approaches, the VHH scaold of PIA-KRASv2-Nb emerged intrinsically
humanized (VH3 family, Hu-mAb score = 1.0), eliminating the need for a posteriori
engineering.
Molecular dynamics simulations, extended up to 10 ns, confirm the persistence
of the binding pose and reveal a three-phase mechanism: rapid anchoring, interface
maturation, and convergence to a stable equilibrium state with 30 residue-residue
contacts. These dynamic metrics are consistent with those observed in successful
therapeutic nanobodies like Caplacizumab or VHH72, consolidating the profile of
PIA-KRASv2-Nb (seed 72) as a pan-mutant therapeutic candidate against
KRAS.
This study demonstrates that the PIA method can generate therapeutically
optimal nanobodies ab initio, combining high affinity, intrinsic humanization, and
conformational reproducibility, with direct implications for tackling targets traditionally
considered "undruggable."