# 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?
developed the inhibitor palmitostatin B against APT1, which perturbated the deacylation cycle of H-Ras in Madin-Darby canine kidney (MDCK)-F3 cells (a Harvey murine sarcoma virus-transformed derivative of MDCK cell line). Inhibition of thioesterase activity over the long-term using Palmitostatin B leads to the loss of steady-state distribution of palmitoylated Ras. However, the study points out the fact that treatment with palmitostatin could also likely affect the spatial distribution of other palmitoylated proteins [90]. Moreover, these strategies would only be effective in H/N-Ras-driven oncogenesis and as K-Ras4B is not palmitoylated, would fail in about 85% of Ras-driven cancers caused due to K-Ras mutation [91]. As palmitoylation is a very dynamic process and its dynamics may vary in normal versus oncogenic cells, the generation of a palmitoylation/acylation inhibitor would require extensive research. This led researchers to design inhibitors against activators-guanine nucleotide exchange factors (GEFs) and effectors of Ras. GEFs and GAPs tightly regulate the activation of Ras isoforms by their association/dissociation to GTP. Son of sevenless (SOS) is one of the prime GEFs with multiple binding sites for Ras. [PERSON] et al. developed a cell-permeable \(\alpha\)-helix peptide that mimics the binding element of SOS, and hence the bound peptide was shown to inhibit SOS-induced activation of Ras and thus extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK-MAPK) signaling in cells [92]. The two known members of the mammalian SOS family-SOS1 and SOS2 share about 70% sequence similarity and have similar expression patterns. As the SOS-induced activation of Ras is a tightly regulated process, mutations in SOS1 or SOS2 can lead to its enhanced activity and hence sustained Ras activation. SOS comprises six domains namely- (\(N\)-terminal histone fold (HF) or the H domain (HD), the Dbl homology domain (DH), the pleckrin homology domain (PH), the helical linker (HL) domain, RAS-exchange motif (REM) domain, the Cdc25 domain, C-terminal proline-rich (PR) GRB2-binding (G) domain) [93]. In the inactivated state, the DH-PH domains allosterically autoinhibit SOS by occupying the Ras-binding site. This autoinhibition of SOS is released upon receptor-activated recruitment of SOS to the plasma membrane. Once recruited to the plasma membrane by PH, SOS is activated by binding of phosphatidic acid (PA) to the HD. Further, the REM anchors Ras-GDP and Cdc25 enables the dissociation of GDP [94]. A negative feedback loop controls the activity of SOS in Ras activation wherein SOS dissociates from its adaptor protein GRB2 upon phosphorylation of its C-terminal Serine/Threonine residues. SOS1 is known to be functionally dominant over SOS2, as shown by knockout (KO) studies. Mice with constitutive knockout for _Sos1_, die in mid-gestation, whereas SOS2 is dispensable in mice for growth and development [95]. Moreover, functional redundancy exists between SOS1 and SOS2, as evident by the early death of SOS1/2 double knock-outs (DKO), whereas single KO of _Sos1\({}^{-/-}\)_ or _Sos2\({}^{-/-}\)_ are viable [96]. SOS mutations lie in either one or all of the above-mentioned six domains. One such asopathy due to SOS mutation is Noonan syndrome (NS) characterized by developmental and cardiac defects. It is primarily an autosomal dominant disorder and based on the genes involved in the Ras signaling pathway that are mutated in NS, it is classified into 12 subtypes. As seen from Table 2 NS4 and NS9 are caused by mutations in SOS1 and SOS2, respectively. In NS4, caused by SOS1 mutation, around 183 mutations have been reported which affect 32 amino acid residues. These residues form various clusters--the first group comprises of {p.(protein) threonine 266 to lysine, p. methionine 269 to arginine/threonine, p.lysine 728 to isoleucine, p.tryptophan 729 to leucine, p.isoleucine 733 to phenylalanine mutations in the DH and REM domains. The residues of this cluster are involved in regulatory autoinhibition (as mentioned above).