Christopher J. Brown

Both mouse double-minute 2 (MDM2), an E3 ubiquitin ligase, and its closely related paralog, MDM4, which lacks E3 activity, play central roles in cellular homeostasis. MDM-linked dysfunction is associated with an increased risk of oncogenesis, primarily through targeting the tumor suppressor protein p53 for ubiquitination and degradation. Recent studies have revealed multifaceted roles of MDM proteins that are p53 independent with implications for their oncogenic properties. This review aims to provide an overview of MDM2 and MDM4, by assessing gene and protein structure and implications for protein–protein interactions and functions in cell and animal physiology. We also explore MDM2 and MDM4 role(s) in angiogenesis, a critical feature of solid tumor growth and progression. Finally, we discuss the current landscape in the development of MDM2 and MDM4 inhibitors for cancer therapy.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these ‘design rules’ to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

Interactions between DAXX and numerous protein interaction partners, along with the autoregulation of a DAXX SUMO Interaction Motif (SIM), can be inhibited by stapled and stitched peptides derived from a minimal DAXX binding peptide found in ATRX.

Editorial: Overcoming the cell membrane to target intracellular oncoproteins

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.

Stapled α-helical peptides can bind to and modulate historically intractable targets while addressing the traditional liabilities associated with peptide therapeutics. However, their pipeline advancement has been impeded by the challenges of identifying peptides with sufficient cellular uptake to engage the target protein while lacking off-target toxicities. Here, we advance the field to arrive at a workflow for identifying advanced stapled peptide lead molecules with on-target in vivo activity with no off-target cell proliferation effects. Specifically, we generated a >350-member library based on ATSP-7041, a stapled peptide Mdm2(X) antagonist with validated on-target cellular effects but with significant off-target activity. Key insights from library analysis include 1) a clear correlation between lipophilicity and permeability, 2) removal of positive charge to avoid off-target toxicities, 3) judicious placement of anionic residues to enhance peptide solubility/behavior, 4) optimization of C-terminal length and helicity to enhance cell activity, 5) optimization of staple type/number to avoid polypharmacology. Incorporation of one or more of these attributes led to molecules with improved in vitro and in vivo activities (up to a >292x improved cell proliferation EC ₅₀ ). A subset of peptides were devoid of off-target cell proliferation effects in cell lines lacking wild-type p53 protein (up to a >3800x on-target index). This latter improvement contrasted with clinical Mdm2 antagonistic molecules. Application of these ‘design rules’ to a distinct Mdm2(X) peptide series resulted in rapid improvement in cellular activity (>150x) and removal of off-target toxicities. Overall, the detailed workflow outlined here should help researchers identify stapled α-helical peptides for therapeutic impact.

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.

Identifying new binding sites and poses that modify biological function are an important step towards drug discovery.

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.