# Peptides That Block RAS-p21 Protein-Induced Cell Transformation
## 2 Anti-Onogenic RAS-p21 Peptides from RAS-p21 Itself
### Use of Overlapping Peptides from RAS-p21 to Target Ras-RAF Interactions  
Since a major signal transduction pathway activated by RAS-p21 involves the binding of RAS-p21 to RAF at the cell membrane, resulting in the downstream activation of MEK and ERK, agents have been devised to block this interaction. As an alternative to using conformational analysis to design xs peptides that could block this interaction as described above, the use of overlapping 12-mer (dodecapeptides) from the RAS-p21 protein was introduced. These peptides were then assayed for their abilities to serve as decoys to RAF, causing the displacement of oncogenic RAS-p21 from binding to RAF [28]. These peptides overlapped from the linear sequence of K-ras by two residues. For example, the first two peptides were residues 1-12 and 11-22, etc. This resulted presumably in the synthesis of eighteen dodecamers that were then bound to a membrane with which activated RAF was incubated, and the binding of RAF to these peptides was assayed via chemiluminescence assay. A contiguous string of peptides from the carboxyl terminus of K-RAS-p21 was found involving residues 169-188, including the CAAX box sequence at the carboxyl terminal end. Interestingly, this domain, called the hypervariable domain of RAS-p21, is the only one for which there are significant differences in the amino acid sequences among the various forms of RAS-p21. This domain is apparently highly flexible since its structure cannot be determined by X-ray crystallography [15]. Since it does contain the CAAX sequence that is farnesylated to enable the membrane attachment of RAS-p21, this peptide may block this attachment. The RAS-p21 169-188 peptide was synthesized with a cell-penetrating peptide or CPP (leader sequence) on its amino terminus, termed Mut3 DPTSh1, the whole sequence being: _VKKKKIAELIKIKMSKDGKKKKKKKKSRTRCTVM_, called Mut3 DPTSh1-Ras, where the italicized sequence is the CPP and the bold sequence is K-ras residues 169-188. This peptide was shown to compete for the binding of ras to RAF in MDA-MB-231 breast cancer cells [28]. Unfortunately, other peptide \"hits\" were found but were not assayed [28]. This consideration would include peptides such RAS-p21 residues 31-42 and 41-52 corresponding to peptide segments from the switch 1 domain that is known to interact with RAF [15]. Results for negative control peptides were not reported. Mut3 DPTSh1-Ras was then assayed for its ability to induce cancer cell death in a number of different cancer cell lines and was found to induce significant apoptosis in MDA-MB-231 breast cancer, SW626 ovarian cancer, SW480 colon cancer and HBCx17 breast cancer cell lines, but only minimally affected the cell viability of HCT-116 and HT-29 colon cancer cell lines, even though they carry K-ras mutations [28]. Peculiarly, though this peptide was shown as causing significant apoptosis of SW480 cells, it was found not to affect viability of these cells in the MTT cell viability assay [28]. This peptide also was found to induce a significant reduction in cell viability of H1650, HBEC wt and HBEC Ras V12, the former two lines being non-small cell lung cancer cell lines that have wild-type ras [28]. These findings suggest that this peptide does not specifically affect mutant ras and may even function in a ras-independent fashion. Mut3 DPTSh1-Ras was also evaluated on human chronic lymphocytic leukemia (CLL) cells from CLL patients as well as peripheral blood mononuclear cells from healthy donors [28]. There was marginal, if any, effect, specifically on CLL cells comparing to normal cells from healthy donors. In addition, there was a baseline 40% of apoptosis observed in normal cells with the treatment of this peptide, suggesting it may be toxic to normal cells [28]. This peptide was further tested in nude mice against hematopoietic tumor cell lines of B-cell origin, such as [PERSON]'s lymphoma (Daudi cell line) and hairy cell leukemia (Jok 1 and 5.3 cell lines), the former of which the study claims is chronic lymphocytic leukemia-like [28].