Enhancing Specific Disruption of Intracellular Protein Complexes by Hydrocarbon Stapled Peptides Using Lipid Based Delivery

Abstract

Linear peptides can mimic and disrupt protein-protein interactions involved in critical cell signaling pathways. Such peptides however are usually protease sensitive and unable to engage with intracellular targets due to lack of membrane permeability. Peptide stapling has been proposed to circumvent these limitations but recent data has suggested that this method does not universally solve the problem of cell entry and can lead to molecules with off target cell lytic properties. To address these issues a library of stapled peptides was synthesized and screened to identify compounds that bound Mdm2 and activated cellular p53. A lead peptide was identified that activated intracellular p53 with negligible nonspecific cytotoxicity, however it still bound serum avidly and only showed a marginal improvement in cellular potency. These hurdles were overcome by successfully identifying a pyridinium-based cationic lipid formulation, which significantly improved the activity of the stapled peptide in a p53 reporter cell line, principally through increased vesicular escape. These studies underscore that stapled peptides, which are cell permeable and target specific, can be identified with rigorous experimental design and that these properties can be improved through use with lipid based formulations. This work should facilitate the clinical translation of stapled peptides.

The MDM2/MDMX-p53 Antagonist PM2 Radiosensitizes Wild-Type p53 Tumors

“…3A and B). Multiple studies have indicated that active transport has a significant role in cellular uptake of stapled peptides, where FAM labeled stapled peptides have been shown to be actively endocytosed into cytoplasmic vesicles (22,23). This is in line with ongoing work in the p53lab on PM2 and other stapled peptides, and with results reported by Yurlova and colleagues (24) demonstrating that inhibition of the MDM2:p53 interaction takes approximately 30 to 60 minutes to occur, as opposed to Figure 4.…”

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function

“…The enriched localization of EGFP-MOP3 in the nucleus via binding to its targets MDM2 and MDMX suggested that treatment with competitive inhibitors could free EGFP-MOP3 to re-equilibrate throughout the cells. We induced expression of EGFP-MOP3 in the stable cell lines and examined the localization of EGFP-MOP3 after treatment with sMTIDE-02, Nutlin or negative control stapled peptide sMTIDE-02 SCRAM ( Thean et al , 2017 ) by timelapse confocal microscopy (Fig. 8 ).…”

Hitting Undruggable Targets: Viewing Stabilized Peptide Development through the Lens of Quantitative Systems Pharmacology

“…Adopted from the viral and gene delivery fields, positive charge is one method of conferring membrane permeability of peptides [6,39,40]. However, methods such as the recombinase enhanced bimolecular luciferase complementation platform (ReBiL) and LDH release quantification highlighted some of the toxicity hurdles inherent in cationic agents and motivated other strategies to achieve membrane permeability [12,38]. Just a few years since the first hydrocarbon stapled peptides were introduced, researchers in the field are developing new membrane permeable peptides with more robust methods of demonstrating on-target efficacy using in vitro assays and giving special attention to physicochemical properties outside of charge that can help improve cytosolic delivery [41][42][43][44].…”

“…However, there are far fewer reports of in vivo efficacy studies, the precursor to clinical trials, and some of the strategies and mechanisms employed towards efficacy in cells may have deleterious effects in the context of an animal model or patient. Furthermore, various studies have revealed uncertainties in how effectively some of these recently developed peptides penetrate cells and bind their targets [11,12]. Efficient cell membrane permeability is just one of many variables to consider for successful in vivo drug delivery.…”