Towards understanding cell penetration by stapled peptides

Chu, Qian; Moellering, Raymond E.; Hilinski, Gerard J.; Kim, Young Woo; Grossmann, Tom N.; Yeh, Johannes T.‐H.; Verdine, Gregory L.

doi:10.1039/c4md00131a

Cited by 197 publications

(334 citation statements)

References 41 publications

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“…Cell penetration is likely to be further enhanced by the overall positive charge carried by SP1, as previous studies have suggested that positive charge is often favorable for uptake of constrained peptides into cells (36). A systematic study of Ͼ200 peptides showed that charges of ϩ1 to ϩ7 are optimal for cellular uptake; as SP1 has a charge of ϩ2, it fits the profile of a peptide that readily enters cells (53). The same study showed that the mechanism of cell uptake of stapled peptides likely involves ATP-dependent endocytosis but is not dependent on caveolin or clathrin.…”

Section: Discussionmentioning

confidence: 87%

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Thomas

Cooper

Clayton

et al. 2016

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 87%

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Thomas

Cooper

Clayton

et al. 2016

Journal of Biological Chemistry

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“…Among the various staple types, the hydrocarbon and triazole staples are among the most stable chemically and the least susceptible to biological degradation. There is ample evidence to suggest that the hydrocarbon and triazole staples enable cell penetration [22,37], which is an important property that allows access to intracellular drug targets. The design principles presented in this review are derived mainly from case studies of these two highly promising classes of stapled peptides, but they can also be applied to the other types of staple.…”

Section: Introductionmentioning

confidence: 99%

Stapled peptide design: principles and roles of computation

Tan

Lane

Verma

2016

Drug Discovery Today

101

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“…They include stapled peptides (1,2), cyclic peptides (3-6), or peptide analogs, such as peptoids (7)(8)(9)(10)(11), that can affect intracellular targets by interfering with protein-protein interactions. The prototypical class of CPPs is the arginine-rich peptides, best represented by R9 or HIV TAT CPPs (12).…”

Section: From the Department Of Biochemistry And Biophysics Texas Aandmentioning

confidence: 99%

Membrane Oxidation Enables the Cytosolic Entry of Polyarginine Cell-penetrating Peptides

Wang

Sun

Muthukrishnan

et al. 2016

Journal of Biological Chemistry

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Arginine-rich peptides can penetrate cells and consequently be used as delivery agents in various cellular applications. The activity of these reagents is often context-dependent, and the parameters that impact cell entry are not fully understood, giving rise to variability and limiting progress toward their usage. Herein, we report that the cytosolic penetration of linear polyarginine peptides is dependent on the oxidation state of the cell. In particular, we find that hypoxia and cellular antioxidants inhibit cell penetration. In contrast, oxidants promote cytosolic cell entry with an efficiency proportional to the level of reactive oxygen species generated within membranes. Moreover, an antibody that binds to oxidized lipids inhibits cell penetration, whereas extracellularly administered pure oxidized lipids enhance peptide transport into cells. Overall, these data indicate that oxidized lipids are capable of mediating the transport of polyarginine peptides across membranes. These data may also explain variability in cell-penetrating peptide performance in different experimental conditions. These new findings therefore provide new opportunities for the rational design of future cell-permeable compounds and for the optimization of delivery protocols.

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Towards understanding cell penetration by stapled peptides

Abstract: A systematic study on cell penetration by stapled peptides.

Cited by 197 publications

References 41 publications

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Stapled peptide design: principles and roles of computation

Membrane Oxidation Enables the Cytosolic Entry of Polyarginine Cell-penetrating Peptides

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