# Ras Isoforms from Lab Benches to Lives--What Are We Missing and How Far Are We?
## 6 Future Recommendations for Devising Isoform-Specific Targeting  
Various attempts have been made in the last four decades to target Ras but most approaches have seen no success. Functional compensation of enzymes is one of the main reasons for the failure of enzymatic targets. For example, geranyl geranyl transferase, functionally compensating for inhibition of protein farnesyl transferases (PFTs). This is exemplified by the functional overlap between Ras isoforms in some specific contexts. Similar approaches to target other enzymes involved in the post-translational machinery of Ras also didn't prove to be promising. It clearly shows our lack of understanding of the complexity of structural and functional specificities of Ras isoforms. The key reason being that the functional diversity of the isoforms was overlooked in all such cases where a general inhibitor was sought after. K-Ras is most frequently mutated at codons 12, 13, and 61, which either perturbs its intrinsic GTPase activity or interferes with GAP-mediated GTP hydrolysis. However, the mechanism of such perturbations is different on mutations at different codons. K-Ras is mutated in about 90% of pancreatic cancers and 98% of these mutations occur at codon 12. The underlying basis of activating mutation at residue 12 is owing to the replacement of glycine with any other amino acid (except for proline), which leads to steric hindrance with the arginine of GAP [158,159]. The most common missense mutations are G12A, G12C, G12D, G12F, G12L, G12R, G12S, G12V [160]. Thus, mutation at codon 12 directly affects the binding and hence the activity of GAP. Biochemical and structural studies have further pin-pointed differences in the G12 mutant in terms of intrinsic GTP hydrolysis and preference in effector protein binding [161,162]. In G12C mutants, the cysteine residue is close to the nucleotide-binding pocket as well as the switch region that binds to effector proteins [163]. Small molecule inhibitors that covalently bind to this cysteine can effectively target G12C mutants. ARS-853 [164] and subsequently an improvised ARS-1620 [165] were developed which showed G12C mutant specific inhibitory activity. Since these inhibitors specifically interact with the thiol group of cysteine, the use is limited to G12C mutants. Currently, GTP analogs that can compete for the binding site of Ras are being developed like the one to target the G12C mutant. These analogs bind near the GTP-binding site or active site or between switch II and helix3. The limitation of these analogs lies in their inability to target the side chain of G12C mutant (the side-chains for mutation at positions 12, 13, and 61 are different) [166]. Another limitation of the K-Ras G12C inhibitor is that amplification of H/N or K-Ras levels in the cell itself can lead to dimerization and hence elevated GTP-bound Ras and thus confer resistance against the inhibitor. Another major challenge that lies in the way of generating a blocking inhibitor is the structural constraints in the structure of Ras isoforms. The structure of Ras is flat and it lacks binding pockets for such inhibitors. Currently, only G12C is targetable because of the presence of a nucleophilic thiol of the cysteine group which can be targeted using covalently binding cysteine-reactive small molecules. [PERSON] et al. identified this new allosteric pocket called S-IIP, which when occupied by a small molecule inhibitor shifts the nucleotide affinity of K-Ras from GTP to GDP state and also reduces its interaction with effectors and regulators [163]. In 2020, Amgen (Thousand Oaks, CA, USA) and Mirati Therapeutics (San Diego, CA, USA) introduced two K-Ras G12C inhibitors, namely AMG 510 [167] and MRTX849 [168]. The effect of these two inhibitors on tumor growth was assessed in 20 cell lines. Both the inhibitors showed mutant-selective inhibition of tumor growth with a marked effect on K-Ras (mutant-G12C)-ERK-MAPK and mTOR-S6K kinase pathways. In vivo studies in tumor-bearing mice fortified the anti-tumor effects of AMG 510 and MRTX849.