RESUMEN
Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.
Asunto(s)
Péptidos de Penetración Celular , Ciclotidas , Neoplasias , Humanos , Ciclotidas/farmacología , Ciclotidas/metabolismo , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismoRESUMEN
DAXX (Death Domain Associated Protein 6) is frequently upregulated in various common cancers, and its suppression has been linked to reduced tumor progression. Consequently, DAXX has gained significant interest as a therapeutic target in such cancers. DAXX is known to function in several critical biological pathways including chromatin remodelling, transcription regulation, and DNA repair. Leveraging structural information, we have designed and developed a novel set of stapled/stitched peptides that specifically target a surface on the N-terminal helical bundle domain of DAXX. This surface serves as the anchor point for binding to multiple interaction partners, such as Rassf1C, p53, Mdm2, and ATRX, as well as for the auto-regulation of the DAXX N-terminal SUMO interaction motif (SIM). Our experiments demonstrate that these peptides effectively bind to and inhibit DAXX with a higher affinity than the known interaction partners. Furthermore, these peptides release the auto-inhibited SIM, enabling it to interact with SUMO-1. Importantly, we have developed stitched peptides that can enter cells, maintaining their intracellular concentrations at nanomolar levels even after 24 hours, without causing any membrane perturbation. Collectively, our findings suggest that these stitched peptides not only serve as valuable tools for probing the molecular interactions of DAXX but also hold potential as precursors to the development of therapeutic interventions.
RESUMEN
Identifying new binding sites and poses that modify biological function are an important step towards drug discovery. We have identified a novel disulphide constrained peptide that interacts with the cap-binding site of eIF4E, an attractive therapeutic target that is commonly overexpressed in many cancers and plays a significant role in initiating a cancer specific protein synthesis program though binding the 5'cap (7'methyl-guanoisine) moiety found on mammalian mRNAs. The use of disulphide constrained peptides to explore intracellular biological targets is limited by their lack of cell permeability and the instability of the disulphide bond in the reducing environment of the cell, loss of which results in abrogation of binding. To overcome these challenges, the cap-binding site interaction motif was placed in a hypervariable loop on an VH domain, and then selections performed to select a molecule that could recapitulate the interaction of the peptide with the target of interest in a process termed Peptide Epitope Linker Evolution (PELE). A novel VH domain was identified that interacted with the eIF4E cap binding site with a nanomolar affinity and that could be intracellularly expressed in mammalian cells. Additionally, it was demonstrated to specifically modulate eIF4E function by decreasing cap-dependent translation and cyclin D1 expression, common effects of eIF4F complex disruption.
RESUMEN
An attractive approach to target intracellular macromolecular interfaces and to model putative drug interactions is to design small high-affinity proteins. Variable domains of the immunoglobulin heavy chain (VH domains) are ideal miniproteins, but their development has been restricted by poor intracellular stability and expression. Here we show that an autonomous and disufhide-free VH domain is suitable for intracellular studies and use it to construct a high-diversity phage display library. Using this library and affinity maturation techniques we identify VH domains with picomolar affinity against eIF4E, a protein commonly hyper-activated in cancer. We demonstrate that these molecules interact with eIF4E at the eIF4G binding site via a distinct structural pose. Intracellular overexpression of these miniproteins reduce cellular proliferation and expression of malignancy-related proteins in cancer cell lines. The linkage of high-diversity in vitro libraries with an intracellularly expressible miniprotein scaffold will facilitate the discovery of VH domains suitable for intracellular applications.
Asunto(s)
Factor 4E Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Técnicas de Visualización de Superficie Celular , Factor 4E Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Biblioteca de Genes , Cadenas Pesadas de Inmunoglobulina/genéticaRESUMEN
Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Supervivencia Celular , Células HEK293 , Humanos , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
In this report we describe the development of a Fluorescent Protein-Protein Interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and Mdm4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and Mdm4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.