Inhibition of Ral GTPases Using a Stapled Peptide Approach

Abstract

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of ␣-helical-stapled peptides that bind selectively to active, GTPbound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.Ras is well established as the most frequently mutated oncogene in human cancer. This small G protein cycles between an active GTP-bound state and an inactive GDP-bound state. Molecular switching between the "on" and "off" states is positively regulated by nucleotide guanine exchange factors (GEFs) 4 and negatively regulated by GTPase-activating proteins (GAPs). It is only the active, GTP-bound form of the protein that can bind to downstream effectors and facilitate signal transduction. Mutations in oncogenic Ras result in a constitutively active protein that remains fixed in the GTP-bound on-state, leading to unregulated activation of downstream pathways. These mutations are found in ϳ30% of all cancers, with a higher occurrence in specific cancer types such as pancreatic (71%) and colorectal (45%) (1). This makes Ras a crucial cancer therapeutic target; nevertheless, Ras has so far evaded direct attempts at inhibition, and many Ras-driven cancers are currently deemed undruggable. Ras signaling has proven difficult to disrupt by small, drug-like molecules because its activation of downstream cascades is accomplished through proteinprotein interactions, which have traditionally been avoided as drug targets due to the large, shallow surfaces involved in protein-protein interfaces (2-4). Likewise, there are no obvious clefts or small molecule binding pockets on Ras, and competitive inhibition with the nucleotide is unfeasible due to the extremely high affinity of GTP binding and its high concentration in the cellular environment (5, 6). Logical attempts to interfere with critical post-translational modifications of Ras, such as inhibition of farnesyltransferase, have also proved unsuccessful (for reviews, see Refs. 1, 6 and 7). More e...

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“…Stapled peptides based on a helix from the RASGEF son of sevenless (SOS) has been used to target RAS. By using the RAL binding domain of RALBP1 as a template, the Mott group designed an a-helical stapled peptide that can bind selectively to active RALB-GTP and block downstream effector signaling of RAL (Thomas et al, 2016). These peptides bind to the switch I and II regions of RAL, which are the sites of interaction with RAL effectors like RALBP1 and regulators like RALGEF.…”