# Peptides That Block RAS-p21 Protein-Induced Cell Transformation
## 3 Anti-RAS-p21 Peptides from Expression Libraries
### Peptides and Small Molecules from Libraries That Bind to Multiple Forms of RAS-p21-Gpp-NHp  
An informatic approach to the design of peptides and small molecules that block RAS-p21-induced cell transformation has been carried out using peptide and small molecule libraries [37]. The X-ray crystal structures of wild-type RAS-p21 and five mutant forms, G12V, G12D, G12C, G13D and Q61H were analyzed for overall similarities in structure and properties. For example, all six forms were computed to have an overall negative surface potential [37]. Then, known anti-cancer peptides from the Cancer PPD database were screened for properties such as high surface positive charge, known strong activity against cancer cell lines and other criteria not listed. A total of 19 peptides were selected for their abilities to dock to specific regions, mainly the P1 loop and the switch 1 and switch 2 domains of the six different RAS-p21 proteins, using commercial servers and programs such as the Haddock server using the PRODIGY program, SwarmDock and FlexPepDock [37]. In the docking procedures for each of the 19 peptides, an \"affinity maturation\" procedure was further performed in which the docking procedure was re-performed on each peptide in which each residue of the peptide was replaced with all other naturally occurring amino acids. Binding energies for final peptide structures bound to wild-type and mutant forms of RAS-p21 were computed with a formula that uses the number of charge-charge, charge-apolar, polar-polar, polar-apolar interactions between peptide and RAS-p21 and (presumably isolated) apolar residues and charged residues on the peptide and RAS-p21 [37]. The interactions between peptide residues and RAS-p21 residue were computed if the distance between any of the heavy atoms of the two residues were within 5.5 A. Since the X-ray structure of a RAS-p21 inhibitory peptide bound to this protein was determined [37], the docking procedure was tested on this peptide to determine if its structure bound to the protein could be computed. The resulting computed structure for this peptide was noticeably different from that of the X-ray structure, although it seems to have been docked near the position of the peptide in the X-ray structure. Concurrently, the ZINC database of small molecules was screened for compounds that had similar structures to GppNHp. This search was further screened by the FAF-Drug4 server that utilizes a number of pharmacological criteria such as absorption, distribution, metabolites, half-lives, toxicities and other attributes and other \"rule of thumb\" criteria. Of7305 structures screened, 474 were used for docking studies against the RAS-p21 protein structures [37]. Of the 19 final peptides, two, called LfcinB (FKCRRWQWRMKK) and Retro (LGGIVSA VKKIVDFLG), were computed to have the lowest binding energies and lowest dissociation constants (Kds) to G12D- and G13D-K-RAS-p21 (in the nM range) both at the P1 loop (mutation site) and the switch 1 and 2 domains, the RAF binding sites. Other peptides were computed to have similar but slightly high binding energies and Kds. The lowest energy docked GTP analogues structure from the ZINC database (Zinc 12502230) was computed to have the highest affinity for both structures [37]. Since the both LfcinB and Retro peptides were computed to bind in different regions of the two mutant RAS-p21 proteins, a new peptide containing both peptides was constructed in which the two peptides were connected with a presumably flexible GGGGS connecting or linker peptide. This constructed peptide, Retro-linker-LfcinB, was further modified to contain more positively charged arginine residues, i.e., FKRRRWQWRRKK for LfcinB and LGRIVSAVKKIVDFLG for Retro (substitutions shown in red), to allow for peptide transport across cell membranes [37]. This peptide and the GTP analogue, Zinc 12502230, were concurrently tested on two pancreatic cancer cell lines, MiaPaCa-2 (homozygous for G12C-RAS-p21) and AsPC-1 (homozygous for G12D-RAS-p21) [37].