Total Chemical Synthesis and Folding of All-l and All-d Variants of Oncogenic KRas(G12V)

Abstract

The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered ‘undruggable’ due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all L- and all D-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-D peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target.

Exceptionally high-affinity Ras binders that remodel its effector domain

“…Alternatively, miniproteins can be prepared using Fmoc ( N -(9-fluorenyl)methoxycarbonyl)–based solid-phase peptide synthesis on low-loading Rink Amide MBHA resin. Double couplings are recommended following arginine and isoleucine residues and throughout the N-terminal arginine- and proline-rich region, and disulfide bonds can be formed selectively using 2,2′-dipyridyldisulfide ( 42 ).…”

Xenoprotein engineering via synthetic libraries

“…Mirror-image peptide and protein-based binding molecules are of longstanding interest in biotechnology (84). However, to date their discovery by mirror-image phage or cell-surface display has required chemical synthesis of the pertinent target molecule (43,85,86), which has restricted the practical size limit of proteins that could realistically be targeted. Direct screening of synthetic MIM libraries is a potential solution to this limitation.…”

Chemical Synthesis of Proteins that cannot be Obtained Recombinantly

“…RFX037.D was also established to have a high stability in mouse, monkey, and human plasma, and has a long in vivo half‐life and a complete lack of immunogenicity in mice . This success exemplifies the power of mirror‐image phage display as an enabling technology for the generation of D‐peptide ligands for proteins associated with diseases like Alzheimer's disease, viral infection, and cancer …”