# Peptides That Block RAS-p21 Protein-Induced Cell Transformation
## 3 Anti-RAS-p21 Peptides from Expression Libraries
### Cyclic Peptides Freeze Oncogenic G12D-RAS-p21 in an Inactive GTP-Bound State  
X-ray crystallographic studies on the G12D-RAS-p21 mutant protein bound to the non-hydrolyzable GTP analogue, GppNHp, indicated that a pocket (called P2) was present between the structure of residues 55-65 of the switch 2 domain and the \(\alpha\)-2 helix involving residues 67-73 and was not present in this RAS-p21 protein bound to GDP [29]. This finding showed that at least this site was \"druggable\" and that possibly peptides or small molecules could be found that would bind to the pocket such that the GTP-bound state of G12D-RAS-p21 would be inactivated and would not interact with the downstream target, RAF. Prior X-ray crystallographic and \({}^{31}\)P-NMR studies on mutant T35S-RAS-p21 bound to GppNHp indicated that there were in fact two bound conformational forms called state 1 and state 2. In state 1, the switch 1 domain is not well-defined crystallographically and does not bind to the RBD of RAF (see above) [30]. In state 2, this domain adopts a conformation that results in the binding of this mutant ras protein to RAF [30]. Thus, the possibility existed that a drug could bind in the pocket area of G12D-RAS-p21-GppNHp such that it would freeze the complex in the state 1 inactive conformation, resulting in the selective inactivation of the G12D-RAS-p21 protein. To this end, a high diversity cDNA expression library of over \(10^{12}\) encoded compounds was employed [29], and a series of cyclic peptides was generated and tested for their abilities to block the binding of a double mutated G12D/T35S-RAS-p21 to the RAF RBD. Peptides that satisfied this condition were excluded if they also bound to the GDP-loaded double mutant protein. This process resulted in the selection of three cyclic dodecapeptides, each with an exocyclic Gly residue labeled as KD1, KD2 and KD3. Of these, KD2, shown in Figure 4, was found to have the highest relative affinity for binding to GppNHp-bound mutant RAS-p21. KD2 strongly inhibited the binding of G12D K-RAS-p21 to the RBD (IC\({}_{50}\) = 12.5 uM) and blocked the SOS-mediated nucleotide exchange of the GDP-loaded protein. On the other hand, KD2 did not block wild-type RAS-p21 charged with GppNHp from binding to the RAF RBD, suggesting that this cyclic peptide will block only oncogenic G12D-RAS-p21 and would therefore not affect normal cells. The X-ray crystal structure of KD2 bound to G12D-RAS-p21-GppNHp has been determined [29] and superimposed on the structures of G12D-RAS-p21-GppNHp and G12D-RAS-p21-GDP. The striking feature of these comparisons is that in the KD2-bound structure, there is a major movement of the \(\alpha\)-2 helix by about 40\({}^{\circ}\) in the switch 2 domain and the peptide binds deeply in this groove between this \(\alpha\)-2 helix and the switch 2 loop [29]. This groove is not seen in any of the X-ray structures of RAS-p21 bound to GppNHp. KD2 makes a number of favorable contacts in the groove with Gly 60 (switch 2), Asp 69 (switch 2 and \(\alpha\)-2 helix) and Gln 99 (\(\alpha\)-3 helix). In addition, the Thr 10 residue of KD2 interacts through a water molecule with Asp 12. Importantly, these changes do not occur in the structure of KD2 bound to G12D-RAS-p21-GDP. The segments involved in the unique groove and the structure of KD2 bound in the grove are shown in Figure 5. While the complete structure of the switch 1 domain involved in the binding of RAS-p21-GppNHp to the RAF RBD could not be determined, this domain was found to undergo a large movement (of up to 25 A) in the KD2-bound protein compared with either the same domain of unliganded G12D-RAS-p21 or G12D-RAS-p21-GDP. Another significant movement was found also in the switch 2 domain (of up to 10 A) [29]. Other cyclic peptides containing substitutions for Thr 11 in KD2 were found to have an enhanced effect on the binding of the peptides to G12D-RAS-p21-GppNHp.