Protein-protein interaction (PPI) is a hot topic in clinical research as protein networking has a major impact in human disease. Such PPIs are potential drugs targets, leading to the need to inhibit/block specific PPIs. While small molecule inhibitors have had some success and reached clinical trials, they have generally failed to address the flat and large nature of PPI surfaces. As a result, larger biologics were developed for PPI surfaces and they have successfully targeted PPIs located outside the cell. However, biologics have low bioavailability and cannot reach intracellular targets. A novel class -hydrocarbon-stapled α-helical peptides that are synthetic mini-proteins locked into their bioactive structure through site-specific introduction of a chemical linker- has shown promise. Stapled peptides show an ability to inhibit intracellular PPIs that previously have been intractable with traditional small molecule or biologics, suggesting that they offer a novel therapeutic modality. In this review, we highlight what stapling adds to natural-mimicking peptides, describe the revolution of synthetic chemistry techniques and how current drug discovery approaches have been adapted to stabilize active peptide conformations, including ring-closing metathesis (RCM), lactamisation, cycloadditions and reversible reactions. We provide an overview on the available stapled peptide high-resolution structures in the protein data bank, with four selected structures discussed in details due to remarkable interactions of their staple with the target surface. We believe that stapled peptides are promising drug candidates and open the doors for peptide therapeutics to reach currently "undruggable" space.

https://pure.rug.nl/ws/files/76392250/Title_and_contents.pdf

Stapled Peptides Inhibitors: A New Window for Target Drug Discovery.

The mouse double minute 4 (MDM4) is emerging from the shadow of its more famous relative MDM2 and is starting to steal the limelight, largely due to its therapeutic possibilities. MDM4 is a vital regulator of the tumor suppressor p53. It restricts p53 transcriptional activity and also, at least in development, facilitates MDM2's E3 ligase activity toward p53. These functions of MDM4 are critical for normal cell function and a proper response to stress. Their importance for proper cell maintenance and proliferation identifies them as a risk for deregulation associated with the uncontrolled growth of cancer. MDM4 tails are vital for its function, where its N-terminus transactivation domain engages p53 and its C-terminus RING domain binds to MDM2. In this review, we highlight recently identified cellular functions of MDM4 and survey emerging therapies directed to correcting its dysregulation in disease.

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The long and the short of it: the MDM4 tail so far.

Drugging the p53 pathway has been a goal for both academics and pharmaceutical companies since the designation of p53 as the 'guardian of the genome'. Through growing understanding of p53 biology, we can see multiple routes for activation of both wild-type p53 function and restoration of mutant p53. In this review, we focus on small molecules that activate wild-type p53 and that do so in a non-genotoxic manner. In particular, we will describe potential approaches to targeting proteins that alter p53 stability and function through posttranslational modification, affect p53's subcellular localization, or target RNA synthesis or the synthesis of ribonucleotides. The plethora of pathways for exploitation of p53, as well as the wide-ranging response to p53 activation, makes it an attractive target for anti-cancer therapy.

Small molecule activators of the p53 response.

BACKGROUND Antimicrobial Peptides (AMPs) are an attractive alternative to traditional small molecule antibiotics as AMPs typically target the bacterial cell membrane. A Trp-rich peptide sequence derived from water buffalo (Bubalus bubalis), BuCATHL4B was previously identified as a broad-spectrum antimicrobial peptide. OBJECTIVE In this work, native Trp residues were replaced with other naturally occurring aromatic amino acids to begin to elucidate the importance of these residues on peptide activity. METHODS Minimal Inhibitory Concentration (MIC) results demonstrated activity against seven strains of bacteria. Membrane and bilayer permeabilization assays were performed to address the role of bilayer disruption in the activity of the peptides. Lipid vesicle binding and quenching experiments were also performed to gain an understanding of how the peptides interacted with lipid bilayers. RESULTS MIC results indicate the original, tryptophan-rich sequence, and the phenylalanine substituted sequences exhibit strong inhibition of bacterial growth. In permeabilization assays, peptides with phenylalanine substitutions have higher levels of membrane permeabilization than those substituted with tyrosine. In addition, one of the two-tyrosine substituted sequence, YWY, behaves most differently in the lowest antimicrobial activity, showing no permeabilization of bacterial membranes. Notably the antimicrobial activity is inherently species dependent, with varying levels of activity against different bacteria. CONCLUSION There appears to be little correlation between membrane permeabilization and activity, indicating these peptides may have additional mechanisms of action beyond membrane disruption. The results also identify two sequences, denoted FFF and YYW, which retain antibacterial activity but have markedly reduced hemolytic activity.

Investigation of the Role of Aromatic Residues in the Antimicrobial Peptide BuCATHL4B

The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein-protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry-based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo.The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic. 

The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth <i>In Vivo</i> and Potentiates Radiotherapy

As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6-125) at 1.66 A resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/Mdm4 antagonism and confirms L98 of Mdm4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/Mdm4 plasticity.

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Jiawei Siau

Papers

Structure-activity studies of Mdm2/Mdm4-binding stapled peptides comprising non-natural amino acids