# Ras Isoforms from Lab Benches to Lives--What Are We Missing and How Far Are We?
## 4 Four Decades of Research and Why Ras Remains Undruggable?
Although FTIs such as lonafarnib, antroquinonol, tipifarnib, BMS-214662, and L778123 [77] showed promising results in preclinical trials, they failed in advanced phases of clinical trials. For example, FTIs showed no antitumor activity in pancreatic and colon cancers. [PERSON] et al. found that in the presence of a potent farnesyl transferase inhibitor, N-Ras and K-Ras proteins in human colon carcinoma cell line DLD-1 underwent an alternative prenylation using the enzyme geranylgeranyl transferase-1(GGTase) [78]. The next approach was to develop inhibitors for dual inhibition of both FTase and GGTase [79]. These inhibitors underwent clinical trials but failed due to their high toxicity and low specificity [80]. Since functional compensation of enzymatic activity is quite plausible, alternative use of statins was considered. This was based on the finding that farnesyl pyrophosphate is an intermediate in both cholesterol synthesis and prenylation. However, the rate constants for squalene synthase (an enzyme involved in cholesterol biosynthesis) and FTase are different, and hence the use of statins inhibited cholesterol biosynthesis to a higher fold when compared to farnesylation of Ras [81]. In addition, the therapeutic doses of Statin did not block the prenylation of Ras [82]. Figure 3: Ras isoforms are attached to the inner leaflet of the plasma membrane. Palmitoylation of residues 181 and 184 in H-Ras and residue 181 in N-Ras makes it hydrophobic and enables membrane attachment. The process of palmitoylation is reversible and depalmitoylated H/N-Ras rapidly shuttles between Golgi and Plasma membrane. Polylysine residues in K-Ras give it hydrophobicity and tether it to the plasma membrane. (PDB structures: H-Ras [73]; K-Ras [74]; N-Ras [75]; NGL (WebGL) viewer [76]. The membrane attachment structure is purely for visualization purposes only. The CAAX processing enzymes and RCE1 and ICMT were lucrative targets for drug discovery. Various studies in mice showed that these two enzymes function in a broad and context-dependent manner. Deficiency of _Icmt_ completely blocked transformation in rodent fibroblast, whereas _Rce1_ did not have any pronounced effects [83]. In a K-RasG12D-driven oncogenic model of myeloproliferation, _Rce1_ deficiency aggravated [84], while _Icmt_ deficiency ameliorated the disease [85]. Contrary to this, in the same K-RasG12D-driven oncogenic model of pancreatic cancer, _Icmt_ deficiency accelerated the disease [86]. Cardiac [87], and retinal toxicity [88] of Rce1 inhibition, and the lacunae that exist in our understanding of the mechanism of these enzymes further limit their use as potential drug targets. In addition to farnesylation, palmitoylation is another PTM that enables membrane targeting of H- and N-Ras. As mentioned earlier, palmitoylation increases the hydrophobicity of H/N-Ras and a cycle of palmitoylation and depalmitoylation also alters Ras's subcellular distribution. Depalmitoylation removes Ras from the membrane and it localizes to the Golgi. Zinc finger DHHC (aspartate-histidine-histidine-cysteine) domain-containing protein 9 (DHHC9) and Golgi complex-associated protein 16 (GCP16) are Ras-specific PATs, that have garnered some interest. Chemical inhibitors like 2-bromopalmitate are extensively used in cell culture to block PATs, but their widespread toxicity limits their use. One study shows that specific inactivation of PAT DHHC9 reduces the leukemogenic potential of N-Ras in mice [89]. Thus, generating specific inhibitors against DHHC9 may seem like a promising approach to overcome the problem of toxicity. However, even in Zdhhc9 KO mice, the palmitoylation of N-Ras was reduced but not abrogated suggesting a compensatory palmitoylation by another closely related PAT DHHC14. Acyl protein thioesterases (APTs) are responsible for depalmitoylating Ras isoforms. Using Protein Structure Similarity Clustering (PSSC), [PERSON] et al.