using-phage-display-to-develop-ubiquitin-variant-modulators-for-e3

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

University of Guelph

Benzyloxycarbonyl-phenylalanyl-alanyl-fluoromethyl ketone (Z-FA-FMK) is a protease inhibitor that has been shown to strongly inhibit mammalian orthoreovirus replication. Here we explore the ability of Z-FA-FMK to inhibit three important yet genetically discrete aquatic fish viruses: chum salmon aquareovirus (CSRV), piscine orthoreovirus (PRV), and the rhabdovirus infectious hematopoietic necrosis virus (IHNV). Z-FA-FMK significantly attenuated CSRV in vitro transcription and infectious yield following low-dose (2-20μM) exposure, yet a relatively high dose (200μM) was required to completely block CSRV replication. For PRV and IHNV, no significant attenuation of in vitro viral transcription was observed following low-dose (2-20μM) exposure; and although high dose (200μM) exposure significantly attenuated both PRV and IHNV transcription, neither was completely inhibited. These transcriptional results were similarly reflected in IHNV infectious titre observed at 7days post exposure. PRV titre is currently undeterminable in vitro; however, in vivo intra-peritoneal injection of PRV into juvenile Atlantic salmon (Salmo salar) in conjunction with 1.5mg/kg Z-FA-FMK did not affect PRV replication as measured by blood associated viral transcripts at 14days post challenge. These results indicate that aquatic ortho- and aqua-reoviruses appear to possess resilience to Z-FA-FMK relative to mammalian orthoreoviruses and suggest that environmental parameters or alternative mechanisms for viral replication may affect the efficacy of Z-FA-FMK as an antireoviral compound. Further, as Z-FA-FMK has been shown to irreversibly inhibit cysteine proteases such as cathepsins B and L in vitro at concentrations of ≤100μM, continued replication of IHNV (and possibly PRV) at 200μM Z-FA-FMK suggests that replication of these viruses can occur in a cathepsin-independent manner whereas CSRV likely requires cathepsins or similar cysteine proteases for successful replication.

Z-FA-FMK demonstrates differential inhibition of aquatic orthoreovirus (PRV), aquareovirus (CSRV), and rhabdovirus (IHNV) replication.

Tumor microenvironments are composed of a myriad of elements, both cellular (immune cells, cancer-associated fibroblasts, mesenchymal stem cells, etc.) and non-cellular (extracellular matrix, cytokines, growth factors, etc.), which collectively provide a permissive environment enabling tumor progression. In this review, we focused on the regulation of tumor microenvironment through ubiquitination. Ubiquitination is a reversible protein post-translational modification that regulates various key biological processes, whereby ubiquitin is attached to substrates through a catalytic cascade coordinated by multiple enzymes, including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases. In contrast, ubiquitin can be removed by deubiquitinases in the process of deubiquitination. Here, we discuss the roles of E3 ligases and deubiquitinases as modulators of both cellular and non-cellular components in tumor microenvironment, providing potential therapeutic targets for cancer therapy. Finally, we introduced several emerging technologies that can be utilized to develop effective therapeutic agents for targeting tumor microenvironment.

Targeting the Ubiquitin Signaling Cascade in Tumor Microenvironment for Cancer Therapy.

Targeted inhibition of misregulated protein-protein interactions (PPIs) has been a promising area of investigation in drug discovery and development for human diseases. However, many constraints remain, including shallow binding surfaces and dynamic conformation changes upon interaction. A particularly challenging aspect is the undesirable off-target effects caused by inherent structural similarity among the protein families. To tackle this problem, phage display has been used to engineer PPIs for high-specificity binders with improved binding affinity and greatly reduced undesirable interactions with closely related proteins. Although general steps of phage display are standardized, library design is highly variable depending on experimental contexts. Here in this review, we examined recent advances in the structure-based combinatorial library design and the advantages and limitations of different approaches. The strategies described here can be explored for other protein-protein interactions and aid in designing new libraries or improving on previous libraries.

Article	Year
Z-FA-FMK demonstrates differential inhibition of aquatic orthoreovirus (PRV), aquareovirus (CSRV), and rhabdovirus (IHNV) replication. Virus research\| PubMed ID: 29174718	2018
Targeting the Ubiquitin Signaling Cascade in Tumor Microenvironment for Cancer Therapy. International journal of molecular sciences\| PubMed ID: 33466790	2021
Recent developments in engineering protein-protein interactions using phage display. Protein engineering, design & selection : PEDS\| PubMed ID: 34117768	2021

Olivia Roscow

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