# Computer-Aided Drug Design Boosts RAS Inhibitor Discovery
## 3 Application of CADD Methods in the Development of RAS Inhibitors
### Molecular Dynamics (MD) Simulation
Based on experimental evidence that the Src-induced dual phosphorylation of KRAS Y32/64 disrupts the GTPase cycle to interfere with RAS downstream signaling [102], MD simulation has revealed the complex process of unphosphorylated or phosphorylated KRAS4B in GAP, SOS, and RAF in the GTP/GDP-bound states. The dual phosphorylation of KRAS4B alters the nucleotide binding site conformations and generates perturbations at the catalytic site, resulting in the expansion of the GDP binding pocket and the latency of the intrinsic hydrolysis of RAS GTP. This has identified RAS phosphorylation as a drug target [103]. As a covalent inhibitor targeting the Switch II allosteric pocket (SII-P) for KRAS\({}^{\text{G12C}}\), AMG-510 (SotoRASib) is the first FDA-approved drug discovered by MD simulation. Using all-atom simulation on a long time scale (75 \(\upmu\)s), in MD simulation, [PERSON] and colleagues [104] found that AMG-510 remains stably bound to SII-P during switch swing, rather than fixing KRAS switches based on crystal structure. With the MD simulation, AMG-510 also explains the interaction with KRAS of PTM [104]. The interaction mechanism and kinetic parameters between KRAS\({}^{\text{G12C}}\) and another covalent inhibitor, ARS-853, were also revealed with molecular docking and MD simulation [105]. There is a challenge that the dimerization or oligomerization of RAS with PTM on membranes can only be resolved with recombinant lipid membranes or nanodiscs [106; 55; 107]. Therefore, MD simulation plays a pioneering role in discovering new potential drug targets and strategies by revealing the interface interaction and energetic information of RAS dimer or other RAS-related pathway proteins at the atomic level [53]. MD simulation is also a robust tool for discovering allosteric binding sites. Allostery can modulate protein structure and activity by binding an effector to an allosteric site instead of the orthosteric site [108]. Therefore, the discovery of allosteric sites [109; 110; 111; 112], the exploration of the allosteric mechanism [113; 114; 115; 116], and the targeting of allosteric sites for drug discovery [117; 118; 119] are of great importance. In combination with the transition pathway generation algorithm and MSM analysis, MD simulation helps to identify several key conformational substates in RAS deactivation hydrolysis and a novel potential allosteric binding site for inhibitors to block downstream signaling effects [99]. NS1 is a peptidomimetic that binds to the variable configuration site of RAS to inhibit RAS dimerization and prevent the abnormal activation of the downstream RAF-MEK-ERK pathway. However, the affinity of NS1 for HRAS is reduced by the HRAS\({}^{\text{R135K}}\) mutation. In a 200-ns MD simulation with dynamic network analysis and investigation of the overall architecture of the allosteric network of HRAS, [PERSON] et al. [120] found that HRAS\({}^{\text{R135K}}\) disrupts most of the key interactions at the interface of the wild-type HRAS-NS1 complex and abrogates the original allosteric regulation. There are studies on the allosteric effects of KRAS as the regulated or the regulator. Using aMD and allosteric pathway analysis, the mechanism of the allosteric activation of PI3K\(\alpha\) by KRAS4B was elucidated to the extent that KRAS4B binding disrupts the interaction between the p110 catalytic subunit and the p85 regulatory subunit of PI3K\(\alpha\). This disruption leads to the exposure of the kinase domain of PI3K\(\alpha\), which facilitates its membrane binding and substrate phosphorylation [114]. [PERSON] et al. propose the mechanism of long-range allosteric regulation from Sprouty-related, EVH1 domain-containing protein 1 (SPRED1) to KRAS in the SPRED1-GAP (NF1)-RAS ternary complex via aMD and MSM analysis. NF1, acting as a scaffold, transfers the allosteric effect from the SPRED1 side to another (KRAS binding site), resulting in the restricted conformational change of the NF1 catalytic center for RAS hydrolysis but steadier KRAS-NF1 binding.