Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives

Abstract

RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.

Keywords: Ras druggable pockets; Ras inhibitors; Ras inhibitory strategies; Ras oncogenes; Ras signaling; anticarcinogenic effect; cancer; natural products.

Figure 1

Diagrammatic representation of the functional cycle, upstream regulators, and downstream effectors of Ras proteins.

Figure 2

Approaches for inhibiting Ras oncoproteins biosynthesis, processing, activity, and signaling. Indirect Ras inhibitory approaches include the interference with different processes: expression of Ras oncogenes, Ras processing and membrane localization, activity of Ras regulators, activity of downstream effectors, Ras-dependent cellular features, and the activity of synthetic lethal interactors. Direct Ras inhibitory approaches include the interference with Ras/GEF interaction and exchange activity, Ras/effector interaction, and Ras dimerization.

Figure 3

Ras proteins cycle. Ras proteins (gray) nucleotide exchange is catalyzed by Ras Guanine nucleotide Exchange Factors (GEFs) such as hSOS1 (in yellow). Ras GTP can assume different conformations due to an allosteric switch that is in the dynamic equilibrium between an “off” state (PDB ID: 5p21) and an “on” state (PDB ID: 3K8Y), both spontaneously and in dependence of Ras GTPase Activating Proteins (GAPs) (such as p120GAP in brown and PDB ID: 1WQ1) or Ras effectors binding. A detail of the “on” conformation (in green) is shown in the insert, superimposed on the “off” conformation: the rearrangement in the position of residues Q61 in switch II and Y32 in switch I is evident. The presence of the two glycines in positions 12 and 13 (represented as spheres) are mandatory to allow this rearrangement, which is fundamental for the catalytical mechanism of GTP hydrolysis. Three of the main effectors of Ras are shown in complex with Ras GTP: Raf Ras-binding domain (RBD) is in light blue (PDB ID: 4G0N), RalGDS Ras-interacting domain (RID) are in pink and purple (PDB ID: 1LFD), and PI3K catalytic subunit is in green (PDB ID: 1HE8). Note that RalGDS binds Ras GTP as a heterotetramer. All of the effectors bind Ras only in the active form due to their higher affinity for the Ras GTP conformation of the effector lobe (dark grey). Ras switch I is in orange, and switch II is in blue. The nucleotide is in green sticks, while Q61 and Y32 are shown as yellow sticks in Ras proteins structures.

Figure 4

Structures of Ras proteins binding to different inhibitors. Switch I is in orange, and switch II in blue. Guanine nucleotide is in dark grey balls. Inhibitors are represented as yellow sticks and their surface as a yellow mesh. (A) KRas^G12C GDP bound to disulfide 6 (PDB ID:4luc) [125], (B) KRas^M72C GDP bound to 2CO7 (PDB ID:5vbm) [149], (C) KRas^G12V GDP bound to DARPinK13 (PDB ID:6h46) [151], (D) KRas GDP bound to cmp 4 (PDB ID:4epv) [131], (E) KRas^G12D GCP bound to DCAI (PDB ID:4dst) (Maurer et al., 2012) [131], (F) KRAS^G12D GCP bound to BI 2852 (PDB ID:6gj8) [139], (G) HRas^T35S GNP bound to Kobe2601 (PDB ID:2lwi) [137], (H) KRas^Q61H GNP bound to Abd-7 (PDB ID:6fa4) [138], and (I) KRas GDP Mb(NS1) (PDB ID:5e95) [152].

Figure 5

Approaches for the identification and development of Ras inhibitors from natural sources. HT: high-throughput.