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.

The transcription factors NF-κB and p53 are key regulators in the genotoxic stress response and are critical for tumor development. Although there is ample evidence for interactions between both networks, a comprehensive understanding of the crosstalk is lacking. Here, we developed a systematic approach to identify potential interactions between the pathways. We perturbed NF-κB signaling by inhibiting IKK2, a critical regulator of NF-κB activity, and monitored the altered response of p53 to genotoxic stress using single cell time lapse microscopy. Fitting subpopulation-specific computational p53 models to this time-resolved single cell data allowed to reproduce in a quantitative manner signaling dynamics and cellular heterogeneity for the unperturbed and perturbed conditions. The approach enabled us to untangle the integrated effects of IKK/ NF-κB perturbation on p53 dynamics and thereby derive potential interactions between both networks. Intriguingly, we find that a simultaneous perturbation of multiple processes is necessary to explain the observed changes in the p53 response. Specifically, we show interference with the activation and degradation of p53 as well as the degradation of Mdm2. Our results highlight the importance of the crosstalk and its potential implications in p53-dependent cellular functions.

Alterations in fear learning/generalization are considered to be relevant mechanisms engendering the development of anxiety disorders being the most prevalent mental disorders. Although anxiety disorders almost exclusively have their first onset in childhood and adolescence, etiological research focuses on adult individuals. In this study, we evaluated findings of a recent meta-analysis of genome-wide association studies in adult anxiety disorders with significant associations of four single nucleotide polymorphisms (SNPs) in a large cohort of 347 healthy children (8–12 years) characterized for dimensional anxiety. We investigated the modulation of anxiety parameters by these SNPs in a discriminative fear conditioning and generalization paradigm in the to-date largest sample of children. Results extended findings of the meta-analysis showing a genomic locus on 2p21 to modulate anxious personality traits and arousal ratings. These SNPs might, thus, serve as susceptibility markers for a shared risk across pathological anxiety, presumably mediated by alterations in arousal.