# Peptides That Block RAS-p21 Protein-Induced Cell Transformation
## 3 Anti-RAS-p21 Peptides from Expression Libraries
### Small Molecules Freeze Oncogenic RAS-p21 in an Inactive GDP Bound State  
Another X-ray structure of RAS-p21 with G12C (CysSH replaces Gly 12) bound to GDP has also been found to have a \"druggable\" pocket or groove in a similar domain to the one described above in Section 3.2 immediately above. This pocket seems to further involve the \(\alpha\)-helix 3 (residues 89-103) [31; 32]. Similar though not identical observations were made for the G12D-K-RAS-p21 mutant [33]. An important feature of this groove is the change in orientation of His 95 in the \(\alpha\)-3 helix, enabling compounds with aromatic rings to enter it. Taking the opposite approach to the one used for cyclic peptides in Section 3.1 above, the strategy in this case was to discover small polycyclic molecules that would bind in this pocket in such a way as to lock G12C RAS-p21 in its GDP-binding state, thereby preventing  
Figure 5: Ribbon representation of the X-ray structure (ref. [29], PDB entry 6 WGN) of KD2 bound to G12D-RAS-p21-GppNHp rotated to show the location of KD2 (pink) in the groove in the switch 2 domain (residues 55–76) which is colored blue except for the \(\alpha\)2 helix involving residues 67–73, colored red. This helix is rotated by 90\({}^{\circ}\) relative to its position in wild-type RAS-p21. The yellow-colored segment is the \(\alpha\)3 helix involving residues 89–103. A significant segment of the pink KD2 peptide is seen to bind in a deep groove between the \(\alpha\)2 helix and a significant part of the switch 2 domain shown as the blue strand to the right and above the pink segment. GppNHp is seen in the middle, right of the figure with the guanine ring on the far right; the \(\beta\)- and \(\Upsilon\)-phosphates (the phosphates are colored orange) are seen to bind to the magnesium ion shown as a green sphere in the center of the figure. The oncogenic Asp 12 residue is shown in the middle of the P loop domain, some of whose other residues (Gly 10 and Ala 11) are also shown. Additionally, to be noted: The all-important switch 1 domain containing residues 32–47 that contacts the RBD of RAF is shown in the upper right of the figure. Some of the residues in this domain such as Pro 34, Ser 39 and Lys 42 are shown to help identify this domain. Between Pro 34 and Ser 39, there is a series of green dashes denoting the absence of location of the atoms in the intervening segment, this is due to the disordered switch 1 domain in state 1 (inactive) in GTP-bound RAS-p21. See text for explanation. it from exchanging GDP for GTP and disenabling it to activate downstream targets such as RAF. Using compounds from small molecule libraries, several compounds were found that have this effect, as shown in Figure 6.  
Of the compounds synthesized and tested, the one with highest affinity, in the picomolar range, for G12C-RAS-p21-GDP was produced at Amgen called AMG510 [34; 35; 36], now called Sotorasib. This compound is a milestone in targeting RAS after being the first anti-RAS therapy approved by the FDA [35]. Its structure is shown in Figure 6A. In the upper left of this figure, it can be seen that an acryl moiety is present that was designed to react covalently with the -SH group of Cys12 in a Michael addition. This compound has been found not only to bind selectively and covalently to G12C-RAS-p21-GDP, but has been found to block the phosphorylation of ERK in a dose-dependent manner, presumably due to its inability to bind to RAF. AMG-510 has been found to enter cells and it induces impaired cell viability in a number of different cancer cell lines including NCI-H358 non-small cell lung carcinoma and MiaPaCa-2 pancreatic cancer cell lines (IC\({}_{50}\) = 6 and 9 nM, respectively). It blocks the viability of most cancer cell lines with the G12C mutation except for a lung alveolar cell carcinoma (SW1573) line. It has also been found to cause a reduction in tumor burden in mice with syngeneic G12C-RAS-p21-induced tumors and has been found to eradicate these tumors in a dose-dependent manner.