Biology subjects

Walport, L. J.

Publications and source records attributed to Walport, L. J..

10 recordsLinked to original sources

bioRxiv2026

Selective binders of a cotranslational folding intermediate

Drug discovery efforts typically target mature, folded proteins, primarily to inhibit their function or induce their degradation. An alternative strategy would be to target incompletely folded states that are transiently populated during protein biogenesis, but whether these states contain druggable pockets was unclear. Here, we used an mRNA display approach to discover cyclic peptides that recognize a nascent polypeptide at an intermediate point during its synthesis on the ribosome. We identified peptides that bind with low-nanomolar affinity to a model nascent protein, {beta}-galactosidase, but do not detectably bind either the fully-folded protein or ribosomes synthesising different sequences. Hydrogen/deuterium exchange-mass spectrometry and cryogenic electron microscopy revealed the molecular basis for selective binding. We found that the nascent chain folds into an -helix against the ribosome surface during ongoing translation, creating a cryptic pocket together with ribosomal proteins uL23 and uL29 that is exploited by the hit peptides. Our results establish proof-of-concept for selective targeting of protein biogenesis intermediates, and provide a platform for future drug development.

biochemistry↗

bioRxiv2026

A potent macrocyclic peptide binder enables effector-free crystallization of the GTPase RAB27A

RAB27A is a member of a highly conserved family of small GTPases that regulate intracellular vesicle trafficking. Multiple studies have linked RAB27A cellular activity to exosome-mediated modulation of the tumour microenvironment and inflammatory signalling, highlighting it as an attractive target for drug discovery. However, identifying structurally validated and selective inhibitors of the RAB27A-effector interaction has proven challenging, partly because wild-type RAB27A crystallizes only in complex with effector proteins such as Slp2a. Using mRNA display, we identified macrocyclic peptide ligands that target the RAB27A effector-binding interface. Optimisation of initial hits led to IMP2660, a nanomolar-affinity RAB27A binder that effectively disrupts RAB27A-effector interactions. IMP2660 enabled determination of a 2.1[A] structure of active-state RAB27A bound to a noncovalent macrocyclic peptide in the absence of an effector, revealing structural features of the effector-binding surface and providing a platform for future structure-guided ligand development.

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↗

bioRxiv2025

Dissecting PADI6 function defines oocyte cytoplasmic lattices as regulatory hubs for fundamental cellular processes

Oocyte cytoplasmic lattices are critical for early embryo development but their composition and function are not fully understood. Mutations in PADI6, an essential component of cytoplasmic lattices, lead to early embryonic developmental arrest and female infertility. To investigate PADI6 function in mRNA storage, global protein levels, and lattice composition during early mammalian development we used single cell transcriptomics and proteomics methods to study two mouse models. Padi6 null mutation resulted in inhibition of embryonic genome activation, defective maternal mRNA degradation, and disruption to protein storage on the cytoplasmic lattices. Distinct developmental phenotypes were observed with a hypomorphic Padi6 mutation. By developing a powerful single cell proteomic fractionation method, we define the cytoplasmic lattice enriched proteome in which we find essential components of another major oocyte-specific compartment, the endolysosomal vesicular assembly (ELVA), suggesting previously unknown interconnections between them. Our findings highlight a critical scaffolding function of PADI6 and implicate cytoplasmic lattices as regulatory hubs for key processes in the oocyte and early embryo, including translation, respiration and protein degradation.

developmental biology↗

bioRxiv2024

Structural Insight into the Function of Human Peptidyl Arginine Deiminase 6

Peptidyl arginine deiminase 6 (PADI6) is vital for early embryonic development in mice and humans, yet its function remains elusive. PADI6 is less conserved than other PADIs and it is currently unknown whether it has a catalytic function. Here we have shown that human PADI6 dimerises like hPADIs 2-4, however, does not bind Ca2+ and is inactive in in vitro assays against standard PADI substrates. By determining the crystal structure of hPADI6, we show that hPADI6 is structured in the absence of Ca2+ where hPADI2 and hPADI4 are not, and the Ca-binding sites are not conserved. Moreover, we show that whilst the key catalytic aspartic acid and histidine residues are structurally conserved, the cysteine is displaced far from the active site centre and the hPADI6 active site pocket appears closed through a unique evolved mechanism in hPADI6, not present in the other PADIs. Taken together, these findings provide insight into how the function of hPADI6 may differ from the other PADIs based on its structure and provides a resource for characterising the damaging effect of clinically significant PADI6 variants.

biochemistry↗

bioRxiv2024

mRNA display in cell lysates enables identification of cyclic peptides targeting the BRD3 extraterminal domain

mRNA display is a powerful technology to screen libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands. These cyclic peptides are particularly well suited to challenging protein targets that have been difficult to drug with small molecules. However, target choice can still be limited as screens are typically performed against purified proteins which often demands the use of isolated domains and precludes the use of aggregation-prone targets. Here, we report a method to perform mRNA display selections in mammalian cell lysates without the need for prior target purification, vastly expanding the potential target scope of mRNA display. We have applied the methodology to identify low to sub-nanomolar peptide binders for two targets; a NanoLuc subunit (LgBiT) and full-length bromodomain-containing protein 3 (BRD3). Our cyclic peptides for BRD3 were found to bind to the extraterminal (ET) domain of BRD3 and the closely related BRD proteins, BRD2 and BRD4. While many chemical probes exist for the bromodomains of BRD proteins, the ET domain is relatively underexplored, making these peptides valuable additions to the BRD toolbox.

biochemistry↗

bioRxiv2023

A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV (PADI4) regulation

Peptidylarginine deiminase IV (PADI4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. Genetic or pharmacological PADI4 inhibition is therapeutically efficacious in mice, but no clinically relevant inhibitors currently exist. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to lack of appropriate systems and tools. Here, we developed and applied a set of potent and selective PADI4 modulators. Using the mRNA- display-based RaPID system, we screened >1012 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, an inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalised for the isolation and study of cellular PADI4; and PADI4_11, a first-in-class activator. Using a newly developed method for the quantification of cellular PADI4 activity, we show that PADI4_3 and PADI4_11 are effective in cells. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work offers new understanding of PADI4 regulation and a toolkit for the study and modulation of PADI4 across physiological and pathophysiological contexts.

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↗

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↗