# 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
These two agents were found to reduce the cell viability of both cell lines by about 60 percent at high micromolar concentrations despite their computed dissociation constants being in the nanomolar range [37]. In view of this fact, it would seem important to determine the binding constants of these peptides to the two RAS-p21 forms experimentally. Additionally, at 200 uM peptide concentration, the cell viability of AsPC-1 cells increased from a little less than 40 percent at 100 uM peptide to over 50 percent at 200 uM peptide. The effect of 100 and 200 uM peptide on the MiaPaCa cells was the same, i.e., the inhibitory effect was the same at about 40 percent cell viability [37]. These results suggest that the agents would leave significant residual tumor in patients treated with them. Surprisingly, these agents were not tested on normal cells to determine if they also inhibited wild-type RAS-p21, especially since the structure of this protein was used in the database peptide screening procedure. As a test for the specificity of the linked peptides and the GTP analogue for inhibiting oncogenic RAS-p21 in cells, their effects on the expression of two downstream targets of RAS-p21, cyclin D and catenin beta-1,encoded by the CCND1 and CTNB1 genes, respectively, were determined [37]. CCND1 expression was reduced in both cell lines, although to a significantly greater extent in MiaPaCa-2 cells. Peculiarly, while these agents almost completely blocked CTNB1 expression in MiaPaCa-2 cells, they increased its expression in the AsPC-1 cells, despite exerting their inhibition of cell viability, suggesting that CTNB1 may not be a reliable marker for ras-21 inhibition [37]. As was performed in the work described above for the KD2 peptide and AMG510, it is desirable to determine if the proposed agents in this study inhibit the binding of oncogenic RAS-p21 to the RBD of RAF oncogenic RAS-p21 and, if so, whether they would also inhibit the binding of wild-type RAS-p21 to the RAF RBD. Use of Cancer Cell-Penetrating Peptides Linked to an Anti-Ras Antibody to Inactivate Oncogenic RAS-p21  
This rather unique approach [38] is based on the discovery that specific cell-penetrating peptides (CPP) appear to be able to enter cancer cells selectively. These peptides have the basic sequence RAGLQFPVGRLLR, called BR1. Two alternate forms contain repeats of the RLLR sequence: BR2 contains one repeat (RAGLQFPVGRLLRRLLR) while BR3 contains two repeats. These three peptides and a Tat CPP control peptide (RKKRQRRR) were each labeled with a FITC fluorophore and were incubated with three cancer cell lines (HeLa, human cervical cancer; HCT116, human colon cancer; and B16/F10 mouse melanoma) and three normal cell lines (HaCat, human keratinocytes; BJ, foreskin fibroblasts; and NIH 3T3, mouse fibroblasts) [38]. Significantly, the BR2 peptide was found to enter all three cancer cells but did not appear to enter any of the three normal control cell lines using immunofluorescence, although flow cytometry indicated about 25-30 percent penetrance (as opposed to 100 percent penetrance for the cancer cells) [38]. The control peptide Tat entered all six-cell lines more or less equally. BR3 showed higher levels of entry for tumor cells but did enter normal cells to a lesser extent while BR1 was found not to enter any of the six cell lines. These results suggested that BR2 enters cancer cells with high selectivity. Further investigation of the specificity of BR2 for cancer cells revealed that the ganglioside synthesis inhibitor, PMPP (1-phenyl-2-palmitoyl-3-morpholino-1-propanol), blocks the entry of BR2 into cancer cells but does not block the entry of the tat peptide into these cells [38]. However, the level of ganglioside expression in the membrane fractions of cancer cells versus that of untransformed cells was not determined.