Fig. 1. Replacement of the KRpep-2d's disulfide bridge with a d-Cys⁵–CH₂–l-Cys¹⁵ thioacetal linkage results in a redox-stable, high affinity peptide. (A) SPR analysis shows that oxidized (cyclic) but not reduced (linear) KRpep-2d binds with high affinity to GDP-loaded KRAS^G12D. The chemical structure is shown, with the N- and C-terminal arginine chains and disulfide linker highlighted in red. (B) Redox-stable peptides MP-6483 and MP-4090 display cell-based inhibition of KRAS signaling (pERK and pAKT) in AsPC-1 cells, as assessed by Western blot at 1 hour and 16 hour time-points; non-binding control enantiomeric peptide MP-4956 showed no activity (Left). Sequences for the peptides are shown. (C) MP-6483 and MP-4090 but not the non-binder controls (MP-9657 and MP-9658) display cell-based inhibition (pERK) in AsPC-1 cells, as assessed by Alpha SureFire Ultra Multiplex Phospho/Total ERK1/2 assay (Perkin Elmer) when treated for 1 h (n = 6, black symbols) and 18 h (n = 2, red symbols). The d-Cys⁵ residue, N- and C-terminal arginine residues, core modifications and thioacetal linker is highlighted in red (D) the same lysates in panel C were also assessed for membrane toxicity, as measured by the CytoTox-ONE™ homogenous membrane integrity assay (Promega). (E) Superimposition of co-crystal structures involving (i) KRpep-2d in complex with KRAS^G12D (GDP) (PDB ID 5XCO, blue) and (ii) MP-9903, a peptide containing the d-Cys⁵–CH₂–l-Cys¹⁵ thioacetal linkage, with KRAS^G12D (GMPPCP) (PDB ID 7ROV, pink), shows a highly similar KRAS conformation and that binding of MP-9903 involves a cis peptide bond between d-Cys⁵ and Pro⁶. For clarity, terminal arginine residues are either transparent or hidden. The d-Cys⁵ residue, N- and C-termini and thioacetal linker are highlighted in red in the structure and the termini changes to MP-6483 is noted.