Drugging the undruggable RAS: Mission possible?

Abstract

Despite more than three decades of intensive effort, no effective pharmacological inhibitors of the RAS oncoproteins have reached the clinic, prompting the widely held perception that RAS proteins are 'undruggable'. However, recent data from the laboratory and the clinic have renewed our hope for the development of RAS-inhibitory molecules. In this Review, we summarize the progress and the promise of five key approaches. Firstly, we focus on the prospects of using direct inhibitors of RAS. Secondly, we address the issue of whether blocking RAS membrane association is a viable approach. Thirdly, we assess the status of targeting RAS downstream effector signalling, which is arguably the most favourable current approach. Fourthly, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, RAS-mediated changes in cell metabolism have recently been described and we discuss whether these changes could be exploited for new therapeutic directions. We conclude with perspectives on how additional complexities, which are not yet fully understood, may affect each of these approaches.

Figure 1. Approaches to discover and develop pharmacologic inhibitors of mutant Ras

Past and ongoing approaches to inhibitors of mutationally activated Ras include Ras-binding small molecules that disrupt a key function(s) of Ras, inhibition of the CAAX motif-targeted enzymes that promote Ras membrane association, inhibitors of effector signalling function, unbiased interfering RNA, genetic or chemical screens for synthetic lethal interactors and inhibitors of metabolism.

Figure 2. Compounds that have been reported to bind to Ras

SCH-53239 was designed to inhibit guanine nucleotide exchange. Structure-activity relationship studies led to the development of a derivative with greater water solubility, SCH-54292. Subsequently, another group used molecular modeling to design a series of sugar-derived bicyclic analogs. Based on earlier observations that the NSAID sulindac showed anti-tumour activity in Hras-mutant rat mammary carcinomas, the active metabolite sulindac sulphide was evaluated and found to bind to H-Ras. IND12 is a sulindac derivative that blocks the growth of Ras-transformed cells^,. MCP1 was identified in a yeast two-hybrid screen for inhibitors of H-Ras binding to full length Raf-1. Zn-cyclen selectively binds and stabilizes the weak effector binding affinity conformational state of Ras. The HBS3 peptide is a mimic of the Sos1 αH helix that interacts with H-Ras. DCAI and VU0460081 were identified in fragment-based library screens for K-Ras4B-binding molecules^,. Kobe0065 was identified in a computer docking screen of a virtual compound library and selected for its ability to inhibit H-Ras-GTP binding to Raf-RBD. Kobe2602 was identified in a subsequent computer-assisted similarity search of 160,000 compounds. A K-Ras G12C inhibitor was identified using a disulphide-fragment-based screening approach with GDP-bound K-Ras G12C. SML-8-73-1 covalently binds to K-Ras G12C and occupies the nucleotide binding site. The Nucleotide exchange activator (compound 4) stimulates Ras-GTP formation, yet disrupts ERK and PI3K signalling.

Figure 3. Three-dimensional structures of Ras/ligand complexes

Ras is represented as a molecular surface, the ligands are in stick models with yellow carbon atoms, and the nucleotide is in stick models with tan carbon atoms. The switch I region is in sky blue, and the switch II region is in purple. a | X-ray structure of a compound covalently linked to K-Ras G12D (PDB 4M22). GCP bound to DCAI (PDB 4DST). b | X-ray structure of K-Ras G12V. GDP bound to VU0460009. c | NMR-derived structure of K-Ras T35S. GNP bound to Kobe 2601 (PDB 2LWI). d | X-ray structure of K-Ras G12C. GDP bound to Shokat compound (PDB 4M22).

Figure 4. Inhibitors of RAS effector signalling under clinical evaluation

Compiled from clinicaltrials.gov. Ras proteins bind to the Ras-binding domain (RBD) of the p110 catalytic subunit of class I PI3Ks (α, γ, and δ). Unless indicated otherwise, PI3K inhibitors are pan-class I. Ras binds to the RBD of A-Raf, B-Raf and C-Raf. mTOR exists as two distinct complexes, mTORC1 (Raptor) and mTORC2 (Rictor). Rapamycin/sirolimus and its analogs (rapalogs: everolimus, ridaforolimus, and temsirolimus) are selective for mTORC1, forming a complex with mTOR and FKBP12. Second-generation mTOR inhibitors are ATP-competitive inhibitors of mTOR kinase activity.

Figure 5. Synthetic lethal interactors in RAS-mutant cancers

Functional screens for mutant RAS synthetic lethal interactors utilize chemically synthesized siRNA libraries or viral vector-based shRNA libraries to identify genes whose knockdown causes selective impairment of the growth of RAS mutant but not RAS WT cell lines. The libraries may be either genome-wide or target a selected set of genes. The library may be delivered well-by-well, as shown in Panel A, or as pooled viruses. RAS synthetic lethal partners may operate in different pathways to support the viability of RAS mutant cells (Panel B). These include co-operating signalling and transcriptional programs, maintenance of genomic stability, and suppression of oncogenic stress.

Figure 6. Ras-driven alterations in metabolism

RAS-mutant cancer cells are characterized by increased macropinocytosis and uptake of albumen, leading to lysosomal degradation and release of amino acids. RAS-mutant cancer cells also exhibit altered autophagy, leading to degradation of organelles and proteins, and to production of amino acids and other components that support metabolism. Oncogenic K-Ras directs glucose metabolism into biosynthetic pathways in PDAC by upregulating many key enzymes in glycolysis. Oncogenic K-Ras induces nonoxidative PPP flux to fuel increased nucleic acid biosynthesis and activates the hexosamine biosynthesis and glycosylation pathways. PDAC cells also utilize a non-canonical pathway to process glutamine and use it to maintain redox status and support growth. Blue text indicates Ras-dependent gene and/or protein expression, with arrows indicating increased (green) or decreased (red) expression. Enzymes are indicated in italics. Abbreviations used are: Glut1, glucose transporter 1; Hk 1/2, hexokinase 1/2; Pfk1, phosphofructokinase 1; Eno1, enolase 1; Pkm, pyruvate kinase; Ldha, lactate dehydrogenase A; Gfpt1, glucosamine-fructose-6-phosphate aminotransferase-1; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; Rpe, ribulose-5-phosphate-3-epimerase; Rpia, ribulose-5-phosphate isomerase; GLUD1, glutamate dehydrogenase 1; GLS1, glutaminase 1; GOT1/2, aspartate transaminase 1/2; MDH1, malate dehydrogenase 1; ME1, malic enzyme; GSH, glutathione; GSSG, glutathione disulfide; ROS, reactive oxygen species.