Biology subjects

Passioura, T.

Publications and source records attributed to Passioura, T..

8 recordsLinked to original sources

bioRxiv2025

De novo Cyclic Peptides Allow Visualisation of the Monomeric and Functional Amyloid Conformations of the Kinase RIPK3

Receptor Interacting Protein Kinase 3 (RIPK3) is a central regulator of necroptosis and a key mediator of inflammatory signaling. Its function is orchestrated through RIP Homotypic Interaction Motif (RHIM)-dependent interactions with other RHIM-containing adapter proteins, forming amyloid-structured necrosomes that trigger kinase activation and subsequently lead to immunogenic cell lysis. Necroptosis eliminates infected or damaged cells and provokes a strong inflammatory response. Dysregulated necroptosis is implicated in chronic inflammatory diseases and associated with ischaemic injury. Despite separate structural elucidation of the isolated RIPK3 kinase domain and the amyloid fibrils formed by the RIPK3 RHIM-containing region, visualizing full-length human RIPK3 in live cells remains challenging due to a lack of specific tools. To address this, we employed Random Non-standard Peptide Integrated Discovery (RaPID) mRNA display to identify cyclic peptides that bind the RHIM-containing region of RIPK3 during its assembly into amyloid fibrils. Three peptides were selected for characterisation and demonstrate utility in visualizing RIPK3 in human cells. These peptides represent promising tools for probing RIPK3 localisation and modulating its function.

biochemistry↗

bioRxiv2025

Peptide molecular glues select between BET paralogues by exploiting allosteric sites and conformational dynamics

Achieving selective target inhibition is critical for minimising drug side effects. This can be especially challenging when targeting individual members of protein families with high sequence similarity. A well-recognised example is the Bromodomain and Extraterminal domain (BET) family of proteins. Chemical inhibition of the acetyllysine (AcK)-binding bromodomains (BDs) of BET proteins has shown considerable promise in a range of disease models. However, despite over a decade of medicinal chemistry efforts, it has proven challenging to develop BET BD inhibitors that exhibit high selectivity between BET family paralogues. Cyclic peptides are versatile scaffolds for therapeutic development and often exhibit high selectivity and affinity for their targets. To explore their potential as selective BET BD inhibitors, we have used mRNA display to identify cyclic peptide ligands for the N-terminal BD of BRD2 and BRD4. The structurally diverse cyclic peptides enriched from the selections boast superior selectivity and affinity to previously developed inhibitors. Most strikingly, we isolated cyclic peptides with [~]1000-fold higher affinity for their target BD over the paralogous BDs, far surpassing selectivities reported to date. Our biochemical and structural data suggest that paralogue-selective cyclic peptides act as molecular glues, exploiting both subtle sequence differences at locations far from the AcK-binding pocket and differences in conformational dynamics between BET BD paralogues to achieve this unprecedented level of specificity. This work provides a blueprint for the development of new classes of selective BET inhibitors and, more generally, underscores the potential of exploiting protein dynamics in the design of selective ligands.

biochemistry↗

bioRxiv2023

mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome

Bromodomains regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given bromodomain. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a bromodomain - the N-terminal bromodomain of BRD3 - using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical bromodomain-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our selected sequences are not found in the human proteome, perhaps suggesting that strong binders to bromodomains might have been selected against.

biochemistry↗

bioRxiv2022

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins

SUMMARYDNA encoded cyclic peptide libraries offer unique opportunities to discover high-potency, high-specificity ligands directed against a target protein. We set out to explore the potential for such libraries to provide ligands that can distinguish between bromodomains from the closely related paralogues of the Bromodomain and ExtraTerminal domain (BET) family of epigenetic regulators. Analysis of peptides isolated from a screen against the C-terminal bromodomain of family member BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, reveals peptides with nanomolar and subnanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved binding features. A subset of the peptides demonstrates significant paralogue-level specificity, though structural analysis does not reveal clear physicochemical explanations for this specificity. Our data demonstrate the power of cyclic peptides to discriminate between highly similar proteins with high potency and hint that differences in conformational dynamics between BET-family bromodomains might modulate binding affinities amongst family members for particular ligands.

biochemistry↗

bioRxiv2021

Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

biochemistry↗

bioRxiv2020

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a {beta}-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.

biochemistry↗

bioRxiv2020

De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinsons disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.

biochemistry↗

bioRxiv2019

Cyclic peptides can engage a single binding pocket through multiple, entirely divergent modes

Cyclic peptide display screening techniques can identify drug leads and biological probes with exceptional affinity and specificity. To date, however, the structural and functional diversity encoded in such peptide libraries remains unexplored. We have used the Random nonstandard Peptide Integrated Discovery (RaPID) system to develop cyclic peptide inhibitors of several acetyllysine-binding bromodomains from the Bromodomain and Extra-Terminal domain (BET) family of epigenetic regulators. These peptides have very high affinities for their targets and exhibit extraordinary selectivity (up to 106-fold), making them the highest-affinity and most specific BET-binding molecules discovered to date. Crystal structures of 13 distinct peptide-bromodomain complexes, which all target the acetyllysine-binding pocket, reveal remarkable diversity in both peptide structure and binding mode, and include both -helical and {beta}-sheet type structures. The peptides can exhibit a high degree of structural pre-organization and bivalent binding of two BDs by one peptide was common, flagging the potential for a new direction in inhibitor design that could bring stronger discrimination between BET-family paralogues. Our data demonstrate for the first time the enormous potential held in these libraries to provide a wide array of modes against a single target, maximizing the opportunity to attain high potency and specificity ligands to a wide variety of proteins.

biochemistry↗