Pharmacological targeting of RAS: Recent success with direct inhibitors

Abstract

RAS has long been viewed as undruggable due to its lack of deep pockets for binding of small molecule inhibitors. However, recent successes in the development of direct RAS inhibitors suggest that the goal of pharmacological inhibition of RAS in patients may soon be realized. This review will discuss the role of RAS in cancer, the approaches used to develop direct RAS inhibitors, and highlight recent successes in the development of novel RAS inhibitory compounds that target different aspects of RAS biochemistry. In particular, this review will discuss the different properties of RAS that have been targeted by various inhibitors including membrane localization, the different activation states of RAS, effector binding, and nucleotide exchange. In addition, this review will highlight the recent success with mutation-specific inhibitors that exploit the unique biochemistry of the RAS(G12C) mutant. Although this mutation in KRAS accounts for 11% of all KRAS mutations in cancer, it is the most prominent KRAS mutant in lung cancer suggesting that G12C-specific inhibitors may provide a new approach for treating the subset of lung cancer patients harboring this mutant allele. Finally, this review will discuss the involvement of dimerization in RAS function and highlight new approaches to inhibit RAS by specifically interfering with RAS:RAS interaction.

Keywords: 3144 (PubChem CID 102004330); ABD7 (PubChem CID 134812710); ARS-1620 (PubChem CID 132274053); BIM-46187 (PubChem CID 11593027); CAAX motif; Cancer; DCAI, PubChem CID 1381961; Deltarasin (PubChem CID 73292904); Effector interaction; GTPase; IND12 (PubChem CID 76715657); Kobe2601 (PubChem CID 163309612); Monobody; Nucleotide exchange; RAS inhibitor; Sulindac sulfide (PubChem CID 5352624); Zn-cyclen (PubChem CID 129651749).

Figure 1.. RAS Proteins.

A) GTPase cycle. Normally, RAS proteins reside in the GDP-bound or inactive state. Following mitogenic stimulation by growth factors, GEFs are recruited to the plasma membrane. Bind of GEFs to RAS results in destabilization in nucleotide binding leading to the release of GDP from RAS and creation of a transient nucleotide free state. Given the high concentration of GTP in cells relative to GDP, RAS proteins load with GTP resulting in the switch to the active state. RAS-GTP recruits and activates it downstream targets such as RAF and PI3K. Termination of RAS signaling occurs through hydrolysis of GTP to GDP. Although RAS possesses intrinsic GTPase activity, it is a poor enzyme. This inactivation step is aided by GTPase accelerating/activating proteins which enhance the GTPase activity of RAS by nearly 100-fold, returning RAS to the inactive, GDP-bound state. B) RAS family members. KRAS4A and KRAS4B are derived from alternative splicing of the same gene resulting in different C-termini. Grey shading highlights residues that are identical in all four RAS proteins. SW1, switch 1 region (aa 30–40); SW2, switch 2 region (aa 60–76); HVR, hypervariable region. Proteins were aligned with Clustal multiple alignment. C) Mutation frequency in RAS alleles. Data were compiled from the Catalogue of Somatic Mutations (COSMIC), v86 [15].

Figure 2.. Distribution of RAS mutations in human tumors.

A) Frequency of mutations in codons 12,13, and 61 in each RAS allele. B) Distribution of specific codon mutations in each RAS isoform. Data were compiled from the Catalogue of Somatic Mutations (COSMIC), v86 [15].

Figure 3.. RAS-inhibitor structures.

Structures of RAS in complex with various inhibitors. Molecular surface of RAS is shaded according to different functional regions: light grey, effector lobe; dark grey, allosteric lobe; SW1, light blue; SW2, dark blue. Ligands are represented as stick models colored yellow. Nucleotide is represented as stick models in tan. The orientations of RAS differ in sets of panels to highlight the binding surfaces of each ligand: Panels A and B are in same orientation; Panels C, D, and E are in same orientation; and Panel F is in a different orientation from all other panels. Additionally, HRAS is shown at a smaller scale in F than depicted in panels A-E to highlight the binding surface of NS1 relative to the total protein. A) KRAS(G12C):ARS1650 (PDB: 5V9U). B) HRAS(M72C):2C07 (PDB: 5VBE). C) HRAS(T35S):Kobe2601 (PDB: 2LWI). D) KRAS(G12D):DCAI (PDB: 4DST). E) KRAS(Q61H):ABD7 (PDB: 6FA4). F) HRAS:NS1 (5E95).

Figure 4.. Proposed model for RAS activation by self-association.

Following mitogen stimulation or oncogenic mutation, RAS becomes loaded with GTP leading to the recruitment of RAF monomers. Two RAS protomers either A) physically interact through specific amino acid side chains or B) move in close enough proximity to facilitate dimerization of downstream targets such as RAF. Binding of NS1 monobody (not shown) in either circumstance would provide significant steric hinderance to prevent RAS-mediated dimerization of RAF, thereby inhibiting RAF activation. Although not depicted in these models, it is possible that RAF is recruited to RAS only after RAS dimerizes, as previously suggested [108].