Direct inhibition of RAS: Quest for the Holy Grail?

Abstract

RAS GTPases (H-, K-, and N-RAS) are the most frequently mutated oncoprotein family in human cancer. However, the relatively smooth surface architecture of RAS and its picomolar affinity for nucleotide have given rise to the assumption that RAS is an "undruggable" target. Recent advancements in drug screening, molecular modeling, and a greater understanding of RAS function have led to a resurgence in efforts to pharmacologically target this challenging foe. This review focuses on the state of the art of RAS inhibition, the approaches taken to achieve this goal, and the challenges of translating these discoveries into viable therapeutics.

Keywords: Cancer; High-throughput screening; RAS biologics; RAS inhibitor; RAS monobody.

Figure 1.. RAS signaling pathways involved in human cancer.

A. RAS cycles between the inactive GDP-bound state and active GTP-bound state. GEFs promote nucleotide exchange whereas GAPs enhance the GTPase activity of RAS returning it to the inactive GDP-bound state. Shown are the five major RAS effect or pathways that play key roles in human cancer. B. Schematic of RAS functional domains. The sequences of the HVRs of the 4 RAS family proteins are shown for comparison. C, Annotated H-RAS crystal structure (PDB 1CRQ) with the allosteric lobe (grey), effector lobe (white), Switch 1 (SW1, yellow) and witch 2 (SW2, tan) regions noted in colors. Regions of alpha helices and beta sheets are marked. GDP is shown in blue.

Figure 2.. RAS prenylation and processing.

(1) ll RAS isoforms are post-translationally modified by farnesyltransferase on the Cys of their CAAX motif. (2) Farnesylated RAS moves to the ER (endoplasmic reticulum) where the AAX is cleaved and the terminal Cys methylated. (3A) H-RAS and N-RAS move to the Golgi and are palmitoylated. (4A) Palmitoylated H-RAS and N-RAS move to the plasma membrane and other endomembranes via the secretory pathway. De-palmitoylation relocalizes H-RAS and N-RAS to the Golgi where they are re-palmitoylated and travel back to the plasma membrane. (3B) K-RAS moves directly to the plasma membrane and other endomembranes through an ill-defined mechanism. (4B) PDEδ dissociates K-RAS from all membranes and relocalizes it to the ER in an ARL-2 dependent manner.

Figure 3.. Oncogenic RAS mutations increase its activity.

A. Wild type RAS is predominantly GDP loaded under basal conditions (left). Oncogenic mutations in codons 12, 13 or 61 reduce enzymatic hydrolysis of GTP to GDP, dramatically increasing the GTP-loaded pool of RAS (right). B. Incidence of specific H-RAS, K-RAS and N-RAS mutations in human cancer. Data acquired from the Catalogue Of Somatic Mutations In Cancer (COSMIC).67 Percentages are based on 1,474 H-RAS, 37,642 K- RAS and 5,092 N-RAS tumor samples.

Figure 4.. Current approaches to inhibiting RAS function.

RAS function has been targeted in a variety of ways such as inhibiting membrane association, targeting nucleotide exchange, disrupting effector interaction and blocking dimerization.

Figure 5.. Binding interfaces of current RAS inhibitors.

Crystal structures of: A. DCAI Bound to K-RAS(WT) (PDB4DST).B. Kobe2601 bound to H-RAS(WT) (PDB 2LWI). C. Shokat’s Compound 12 bound to K-RAS(G12C) (PDB 4M22). D. K27 DARPin bound to K-RAS(WT) (PDB 5O2S). E. Sso7d-based R11.1.6 bound to K-RAS(G12D) (PDB 5UFQ). F. NS1 monobody bound to H-RAS(WT) (PDB 5E95). Coloring scheme is same as in Fig. 1C except inhibitors are shown in red. Orientation of the RAS proteins is identical in each panel.

Figure 6.. Proposed model of RAS activation through dimerization.

RAS cycles between GTP-and GDP-loaded states through the action of GEFs and GAPs. Interaction of RAF with GTP-loaded RAS is not sufficient for activation. Co-association of RAS proteins promotes RAF dimer formation and activation.^,