# Computer-Aided Drug Design Boosts RAS Inhibitor Discovery
## 2 Biochemical Features of RAS  
### RAS in Normal Physiological Condition  
RAS family proteins are encoded by three genes, namely HRAS, NRAS, and KRAS, which have highly homologous sequences and overlapping functions [14]. Among them, KRAS has splice variants representing two isoforms, KRAS4A and KRAS4B. Residues 1-166 form the G domain, which consists of six \(\beta\)-strands (81-86) surrounded by five \(\alpha\)-helices (\(\alpha\)1-\(\alpha\)5) and ten connecting loops (L1-L10). Further structural studies at RAS have revealed P-loop (P or L1; residues 10-17), Switch I (residues 30-38), and Switch II (residues 59-76) on the surface of the G domain, which constitutes the active site for GTP/GDP and serves as interfaces for the binding of effector proteins and regulatory factors (Figure 1a) [15]. Most proteins in the superfamily of P-loop nucleoside triphosphate hydrolases (NTPases) contain a highly conserved sequence motif, P-loop. The P-loop keeps the GTP in an appropriate configuration for nucleophilic attack by a water molecule interacting with the Switch I and Switch II regions [16]. Twenty residues of RAS C-terminal (residues 167-188/189) form a hypervariable region (HVR).  
RAS is a GTPase switch between the GTP-bound active state and the GDP-bound inactive state in several key signaling pathways that regulate cell growth, proliferation, and differentiation (Figure 1b) [17]. This switch is modulated by GTP-activated proteins (GAP) such as p120 GAP, neurofibromin, type I neurofibromatosisis (NF1) gene products, etc. [18] They facilitate GTP hydrolysis. There is a highly conserved \"arginine finger\" on GAP. Once this \"arginine finger\" moves into the active site of RAS-GTP, the intrinsic GTPase activity of RAS is greatly increased. RAS is also modulated by GMP exchange factors (GEF), such as the son of seven less homologue (SOS). They catalyze the loading of GTP to active RAS [19]. Activated RAS anchors to the cell membrane via the C-terminal CAAX box (C, cysteine; A, aliphatic amino acid; X, any amino acid) of the HVR region [20], which is post-translated by farnesyltransferase (FTase) or geranylferanyltransferase (GGTase) for prenylation to dimerize or assemble \"nanoclusters\" of 5 to 10 monomers to regulate downstream signaling [21; 22]. Following isoprene conversion at the CAAX cysteine, the RAS-converting enzyme (RCE1) performs proteolytic cleavage at the RAS AAX terminal, followed by carboxymethylylation by isoprenylcysteine carboxyl methyltransferase (ICMT).  
Figure 1: The RAS functions as a binary switch in normal state. (**a**) Cartoon representation of the crystal structure of RAS complexes: KRAS4B–GTP (modified from KRAS4B–GppNHp; PDB ID: 3 GFT) and KRAS4B–GDP (PDB ID: 4 LPK). The helices (\(\alpha\)1–\(\alpha\)5), strands (\(\beta\)1–\(\beta\)6), and loops (L1–L10) are shown in red, yellow, and gray, respectively. The P-loop (P or L1), Switch I, and Switch II regions are colored lime, pink, and blue, respectively. GTP/GDP are depicted by stick models [23]. (**b**) Schematic diagram showing the RAS-related signaling pathways. After activation by epidermal growth factor (EGF), the tyrosine kinase receptor EGFR recruits GEF such as SOS to the cell membrane via Src homology 2 domain containing (SHC) and growth-factor-receptor-bound protein 2 (Grb2) to activate RAS [24]. Subsequently, the activated RAS dimerizes and binds to the downstream RAF protein to regulate the MAPK signaling pathway (RAS-RAF–MEK–ERK pathway). The activated ERK is transported to the nucleus and then phosphorylates a number of transcription factors, such as erythroblastosis virus transcription factor (ETS), to ultimately regulate the cell cycle [25]. In another RAS-PI3K–AKT pathway, the activated RAS recruits PI3K to phosphorylate the substrate PIP2 and generate PIP3, whereupon PIP3 causes the sequential phosphorylation of AKT and mTOR to regulate cell proliferation [26].