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Targeting Ras with Macromolecules Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Targeting Ras with Macromolecules Dehua Pei, Kuangyu Chen, and Hui Liao Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210 Correspondence: [email protected] Activating Ras mutations are associated with 30% of all human cancers and the four Ras isoforms are highly attractive targets for anticancer drug discovery. However, Ras proteins are challenging targets for conventional drug discovery because they function through intracel- lular protein–protein interactions and their surfaces lack major pockets for small molecules to bind. Over the past few years, researchers have explored a variety of approaches and modalities, with the aim of specifically targeting oncogenic Ras mutants for anticancer treatment. This perspective will provide an overview of the efforts on developing “macro- molecular” inhibitors against Ras proteins, including peptides, macrocycles, antibodies, nonimmunoglobulin proteins, and nucleic acids. as is a small GTPase, acting as a molecular Mutations in K-Ras are particularly prevalent in Rswitch in many signaling pathways and some of the most deadly cancers, including pan- regulating cell proliferation, differentiation, creatic (90% prevalence), colon (35% preva- and survival, among other functions (Young lence), and lung cancers (16% prevalence). Dis- et al. 2009). Its four isoforms, H-Ras, N-Ras, ruption of Ras function genetically (i.e., by gene K-Ras4A, and K-Ras4B, are identical within mutations or small-interfering RNA [siRNA]) the amino-terminal 85 amino acids and differ inhibits the proliferation of Ras-mutant cancer primarily in the carboxyl-termini (amino acids cells and induces apoptosis, validating Ras as 165–189). Wild-type Ras oscillates between the one of the most compelling cancer drug targets inactive guanosine diphosphate (GDP)-bound (Gupta et al. 2007; Singh et al. 2009; Castellano form (Ras-GDP) and the active guanosine tri- et al. 2013; Khvalevsky et al. 2013). phosphate (GTP)-bound form (Ras-GTP) (Fig. Over the past three decades, researchers 1A). Ras-GTP interacts with and activates mul- have explored several approaches to inhibit www.perspectivesinmedicine.org tiple effector proteins, including kinases Raf and Ras function (Wang et al. 2012; Spiegel et al. phosphoinositide 3-kinase (PI3K), turning cells 2014; Stephen et al. 2014). The ideal approach on for proliferation and survival. Somatic mu- would be direct binding to the GTPase active tations at Gly-12, Gly-13, or Gln-61, which are site by a small molecule. However, this approach all located within the GTPase active site, impair was quickly found to be impractical, because GTP hydrolysis, resulting in an excessive Ras- Ras binds to GTP (and GDP) with pM affinity GTP population leading to uncontrolled cell and GTP is present in the cytosol at mM growth. Ras mutations are found in 30% of concentrations. The next-best option is to block all human cancers and are well-established can- the interaction between Ras and its partner pro- cer drivers (Prior et al. 2012; Singh et al. 2015). teins and/or its nucleotide exchange activity. Editors: Linda VanAelst, Julian Downward, and Frank McCormick Additional Perspectives on Ras and Cancer in the 21st Century available at www.perspectivesinmedicine.org Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031476 1 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press D. Pei et al. ABSwitch I Inactive effector-binding site RAS GDP GTP GAP Nucleotide GEF Switch II hydrolysis exchange Active RAS Effector GTP (Raf/PI3K) Dimerization interface Cell proliferation/survival Figure 1. Ras structure and function. (A) Interconversion between inactive Ras-guanosine diphosphate (GDP) and active Ras-guanosine triphosphate (GTP) mediated by guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP). The active Ras-GTP interacts with effector proteins and activates downstream cell signaling. (B) Structure of K-Ras bound with GppNp, a nonhydrolyzable analog of GTP. The switch I/II regions are shown in red, the dimerization interface in blue, and the bound nucleotide analog is shown as sticks. (Figure was recreated from PDB data [pdb5p21].) Unfortunately, Ras has been a very challenging and provides an opportunity for selective inhi- target for direct inhibition, because its surface bition of Ras-mutant cancers. Development has no obvious pocket for small molecules of direct Ras inhibitors is challenging because to bind. Consequently, most of the early efforts Ras performs its biological functions by engag- were focused on targeting the signaling steps ing in intracellular protein–protein interac- upstream or downstream of Ras. Small-molecule tions (PPIs). PPIs are in general a challenging inhibitors against the posttranslational modifi- class of drug targets for conventional small mol- cation of Ras proteins (e.g., inhibitors against ecules (defined as molecules of 500 molecular farnesyl and geranylgeranyl transferases) ulti- weight), because small molecules typically do mately proved to be ineffective clinically, because not make a sufficient number of contacts with of either a lack of efficacy or unacceptable toxic- the large flat PPI interfaces to impart high af- ity (Whyte et al. 1997; Konstantinopoulos et al. finity or specificity. To effectively compete with 2007; Berndt et al. 2011). Another indirect ap- the protein partners, a PPI inhibitor should also www.perspectivesinmedicine.org proach is inhibition of the protein kinases down- possess a binding surface comparable in size to stream of Ras. Several MEK and PI3K inhibitors that of the partner proteins. Recognizing this, are currently undergoing clinical evaluation, al- researchers have increasingly turned their atten- though the clinical effectiveness of this approach tion to larger molecules, including peptides, remains to be seen (Engelman et al. 2008). It macrocycles, and proteins, as PPI inhibitors. appears that simultaneous inhibition of at least Powerful combinatorial library technologies two different pathways (e.g., by both MEK and for discovering peptide—especially macrocyclic PI3K inhibitors) is necessary to effectively block peptide—ligands and novel approaches to Ras signaling in Ras-mutant tumors (Britten improving their membrane permeability have et al. 2013); however, blocking multiple signal- recently been developed (Dougherty et al. ing pathways may result in extensive toxicity. 2017). Application of these technologies to Driven by both urgent unmet medical needs Ras has resulted in a variety of Ras inhibitors, and remarkable technological breakthroughs, ranging from small molecules, peptides, macro- there has been a recent “renaissance” of the di- cycles, proteins, to nucleic acids over the past rect Ras inhibition approach (Ledford 2015), few years (Fig. 2). In this review, we provide a which attacks Ras-driven cancers at the source summary of the “macromolecular” approaches 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031476 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Macromolecular Ras Inhibitors A B O O OONH2 O H H H CI OH N N N N RRFFGI N N LKTEEGN-NH2 N H H I N OO H OH O C D NH2 F N H2N O O H O N NH NH2 O N NH H O O O NH NH O H O N O N H O H N NH N H N N O H H2N OH N N NH NH2 2 H N 2 NH2 NH2 EFG VH VL www.perspectivesinmedicine.org Figure 2. Molecular modalities that have been used as direct Ras inhibitors. (A) Small molecules (example shown is the K-RasG12C-selective inhibitor by Ostrem et al. 2013); (B) stapled peptides (example shown is SAH-SOS1); (C) macrocyclic peptides (example shown is cyclorasin 9A5); (D) monoclonal antibodies; (E) intrabodies (the VH and VL regions of an antibody linked by a polypeptide or disulfide bond); (F) affibodies; and (G) monobodies (the tenth fibronectin type III domain of human fibronectin). to Ras inhibitor development (as opposed to quences of several Ras effector proteins and the small-molecule approach). For an updated identified a consensus Ras-binding sequence account on small-molecule Ras inhibitors, shared among a subset of the Ras effectors. readers are referred to Zhou et al. (2017). They showed that peptides containing this se- quence from Raf-1 (Table 1, compound 1) and PEPTIDE-BASED INHIBITORS NF1-GAP (Table 1, compound 2) blocked NF1- GAP stimulation of Ras GTPase activity and Linear Peptides Ras-mediated activation of mitogen-activated The first peptidyl Ras inhibitors were discovered protein kinases, with IC50 values of 45 mM. by Clark et al. (1996), who compared the se- Similarly, Barnard et al. (1998) systematically Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031476 3 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press D. Pei et al. tested short peptides corresponding to different cell-permeable and reduced epidermal growth regions of H-Ras and Raf-1 for in vitro inhibi- factor (EGF)-stimulated Ras activation and tion of the Ras-Raf association and found that downstream signaling events in HeLa cells. two of the Raf-1 peptides, E94CCAVFR100 (Table More recently, Leshchiner et al. (2015) designed 1, compound 3) and C95CAVFRL101 (Table 1, a hydrocarbon-stapled a-helical peptide with compound 4), were significant inhibitors of much greater Ras-binding affinity, SAH-SOS1 95 101 Ras-Raf binding. Peptide C CAVFRL had (KD ,200 nM) (Table 1, compound 9 and an IC50 value of 7 mM and also inhibited Ras- Fig. 2B), also corresponding to the Ras-binding RalGDS binding. Xu and Luo (2002) screened helical motif in SOS. SAH-SOS1 was insensitive 3.5 Â 107 peptide aptamers and isolated two to the nucleotide-bound states of Ras.
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