# 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? Despite extensive research and understanding of Ras from the last four decades, Ras remains undruggable. The answer lies in the fact that the prerequisite for targeting a molecule for drug discovery requires an in-depth understanding of various nuances of its functioning. Initial studies and attempts to generate an inhibitor to block K-Ras undermined the isoform specificity. Owing to their similarity in structure, H-Ras was considered as the prototype of all the three isoforms, and the initial drug development process primarily focused on the structure of H-Ras. [PERSON] et al. first deciphered the X-ray crystallographic structure of human cH-Ras at a resolution of 2.7 A. An intact protein of 189 residues, a catalytic domain of 1-171 residues with GTPase activity, catalytic domains activated at valine 12 and leucine 21, and their corresponding intact protein were crystalized [67]. Using various slowly hydrolyzing GTP analogs, [PERSON] et al. further provided a more refined crystal structure of guanine nucleotide-binding domain of p21 Ha-ras in its active GTP-bound conformation [68]. Using computational analysis, binding pockets for small molecular inhibitor was sought after, but the problem arose as later discoveries pointed out the absence of deep hydrophobic pockets on the surface of K-Ras. As Ras, a cytosolic protein, undergoes a series of elaborate post-translational modifications (PTM), to be finally targeted to the inner leaflet of the plasma membrane or other compartments, the next approach to target Ras was to develop post-translational modification inhibitors. The C-terminal CAAX motif (C = cysteine, A = an aliphatic amino acid, and X = serine or methionine) first undergoes farnesylation or prenylation with the addition of a 15-carbon farnesyl isoprenoid to the cysteine of CAAX motif by the enzyme farnesyltrans-ferase (FTase). This prenylated protein is inserted into the membrane of the Endoplasmic reticulum (ER). Here, the CAAX motif is cleaved, AAX removed by Ras converting endopeptidaseases (RCE1). The next step is carboxymethyl of the farnesylated cysteine by isoprenylcysteine carboxyl methyltransferase (ICMT). H-Ras and N-Ras undergo another addition of a secondary signal in the Golgi in the form of palmitoylation for its membrane targeting. Herein a 16-carbon palmitoyl chain is added to the cysteine upstream of the farnesylated carboxterminal cysteine by a class of enzymes called palmitoyl acyltransferases (PAT). Palmitoylation occurs at cysteine 181 and 184 for H-Ras and at residue 181 for N-Ras, which makes these isoforms highly hydrophobic. Genetic studies in yeast have shown that Erf4p, a Ras modification peripheral membrane protein of ER, in complex with an integral membrane protein Erf2p, is required for palmitoylation as well as non-classical membrane targeting of Ras proteins [69]. K-Ras follows a Golgi-independent targeting to the plasma membrane [70], where it is tethered to the cytoplasmic face. K-Ras is tethered to the negatively charged phospholipids of the inner leaflet by its polybasic lysine region, which is positively charged (Figure 3) [71]. Myristoylated alanine-rich C kinase substrate (MARCKS) is a protein that binds to the PM. Protein kinase C (PKC)-mediated phosphorylation of serine residues in the polybasic region causes neutralization of charges and hence MARCKS dissociates from the PM [72]. The association of Ras with the membrane is imperative to its functioning. Cyclic events of palmitoylation by PATs and depalmitoylation by acyl protein thioesterase 1 (APT1) lead to the redistribution of palmitoylatable Ras from the membrane back to the Golgi. Farnesylation is a process that all the three Ras isoforms undergo; hence farnesyl transferase inhibitors were branded as the next cancer drug. Peptide and non-peptide mimetics generated from the CAAX template that served as an alternate substrate for FTase, other thiols, and non-thiol FTIs were developed subsequently.