Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells

Abstract

Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein−protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT−KD3 and cR10−KD3, able to activate the p53 pathway in cancer cells. cTAT−KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1−12 μM. cR10−KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 μM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors

“…Differences in binding selectivity were also previously observed for optimized stapled KD3-peptides with picomolar affinity for MDM2, and ∼1 nM activity for MDM4. 15 Due to the presence of hydrophobic pockets in the p53 binding site on MDM2, we speculated that the two biphenyl moieties may form key pharmacophores for the high binding affinity observed. Indeed, design of an inactive mutant was achieved by exchanging the biphenyl amino acid in position 11 with an Ala.…”

“… 13 Along those lines, very recently it was demonstrated that conjugation of cCPPs via triazole linkages to the peptide KD3, which was known to be a non-cell permeable stapled peptide inhibitor of p53/MDM2 and p53/MDMX interactions, 14 enables activation of apoptosis in cancer cells. 15 Alternative approaches for creating cell-permeable peptides to disrupt p53/MDMX interactions using cyclic peptide scaffolds, 16 polycationic tags or medchem optimization strategies have been extensively studied. 17,18 …”

Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

“…To increase the penetration into cells, the peptide was modified by attaching lysine residues to the C- and N-terminus, which made it possible to enhance its p53-dependent anticancer activity [ 81 ]. On the basis of another peptide inhibitor, KD3, conjugates with a cell-penetrating peptide (cTAT-KD3 and cR10-KD3) were designed, which were able to activate the p53-dependent signaling pathway in cell models when exposed to a lower micromolar range [ 82 ]. Peptidomimetics with extremely high resistance to proteolysis in vivo based on right-handed alpha-helical sulfono-γ-AA peptides inhibited the complex formation of MDM2/p53 and MDMX/p53 by binding to MDM2 and MDMX with K D values of 19 and 67 nM, respectively.…”

“…It is interesting to note that this construct had much higher in vitro activity than the reference low molecular weight compound Nutlin-3 (Mr = 582, CAS No, 890090-75-2) [ 84 ]. It should be noted that studies [ 81 , 82 , 83 , 84 ] were focused more on achieving high binding affinity of interfacial peptides and optimizing cell penetration, but not on achieving specificity of delivery to tumor cells. However, a study by Lundsten and co-authors [ 85 ] addressed this problem.…”

Antimicrobial peptides provide wider coverage for targeting drug‐resistant bacterial pathogens

“…For this reason, endosomal escape is recognised as the rate-limiting step for the efficiency of CPPs against intracellular targets. [129,200] However, promising examples of CPP-peptide conjugates [201] and antimicrobial CPPs with modified sequence [141] have been shown to reach the cytosol of cells, to act on intracellular targets, by escaping endosomes (e.g., cTAT-KD3) [201] and/or direct translocation of the cell membrane (e.g., cR10-KD3). [201] Of great importance, the modified antimicrobial CPP [I11S]tachyplesin I can reach bacteria that are sequestered inside vesicles within host macrophages, resulting in significant reduction in bacterial load without damaging the host cell membranes.…”