Biology subjects

Wilkinson-White, L.

Publications and source records attributed to Wilkinson-White, L..

4 recordsLinked to original sources

bioRxiv2026

Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

Chemical modifications such as 2'-O-methyl (2'-OMe) are central to the efficacy and tolerability of RNA therapeutics. We recently identified that 2'-OMe RNA fragments as short as three nucleotides can exert opposing effects on Toll-like receptor 8 (TLR8) sensing in a motif-dependent manner. This discovery raises important considerations for degradation products of chemically modified RNA therapeutics, which may generate such immunologically active fragments. Here, leveraging their short length, we systematically map how base and sugar modifications within 3-mer oligonucleotides regulate TLR7 and TLR8 responses, and we resolve the structural basis for both TLR8 potentiation and TLR7/8 antagonism by RNA fragments. Building on these insights, we report the development of a dual TLR7/8 inhibitory oligonucleotide with therapeutic potential in autoimmune disease. Together, these findings provide unprecedented resolution of the immunomodulatory properties of oligonucleotide modifications on TLR7/8 and establish 3-mer oligonucleotides as the shortest functional class of RNA therapeutics described to date.

immunology↗

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↗

bioRxiv2024

2'-O-Methyl-guanosine 3-base RNA fragments mediate essential natural TLR7/8 antagonism

Recognition of RNA fragments by Toll-like receptors (TLR) 7 and 8 is a key contributor to the initiation of a protective innate immune response against pathogens. A long-standing enigma is how degradation products of host RNAs, generated by the daily phagocytic clearance of billions of apoptotic cells, fail to activate TLR7 and TLR8 signalling1. Here, we report that select 2-O-methyl (2-Ome) guanosine RNA fragments as short as 3 bases, including those derived from host-RNAs, are potent TLR7 and TLR8 antagonists that reduce TLR7 sensing in vivo. Mechanistically, antagonistic fragments are directed towards a distinct binding site on these proteins by 5-end 2-Ome guanosine. Our results indicate that host-RNAs evade detection by TLR7/8 due to a pool of abundant host ribosomal 2-Ome-modified RNA fragments that naturally antagonize TLR7 and TLR8 sensing to avoid auto-immunity. Crucially, rare TLR7 and TLR8 mutations located at this antagonistic site decrease the inhibitory activity of 2-Ome guanosine RNA fragments and lead to auto-immunity in patients. Our findings also establish that select chemically synthesised 3-base oligonucleotides can harness the protective anti-inflammatory activity of this natural immune checkpoint for therapeutic targeting of TLR7-driven diseases. One Sentence SummaryShort 2-O-Methyl RNA fragments are natural TLR7/8 antagonists

immunology↗

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↗