A cell permeable bimane-constrained PCNA-interacting peptide

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 3₁₀-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with K(D) values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 3₁₀-helical binding conformation. This suggests the 3₁₀-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.Aimee J. Horsfall, Beth A. Vandborg, Zoya Kikhtyak, Denis B. Scanlon, Wayne D. Tilley, Theresa E. Hickey, John B. Bruning and Andrew D. Abel

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Targeting PCNA with peptide mimetics for therapeutic purposes

Proliferating cell nuclear antigen (PCNA) is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad‐spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA‐interacting protein (PIP) box. There is large sequence diversity between high‐affinity PCNA binding partners, but with conservation of the binding structure - a well‐defined 3₁₀‐helix. Herein, all current PIP‐box peptides crystallised with human PCNA are collated to reveal common trends between binding structure and affinity. Key intra‐ and intermolecular hydrogen‐bonding networks that stabilise the 3₁₀‐helix of PIP‐box partners are highlighted and related back to the canonical PIP‐box motif. High correlation with the canonical PIP‐box sequence does not directly afford high affinity. Instead, we summarise key interactions that stabilise the binding structure that leads to enhanced PCNA binding affinity. These interactions also implicate the “non‐conserved” residues within the PIP‐box that have previously been overlooked. Such insights will allow a more directed approach to develop therapeutic PCNA inhibitors.Aimee J. Horsfall, Andrew D. Abell and John B. Brunin

A turn-on fluorescent PCNA sensor

The solvatochromic amino-acids 4-DMNA or 4-DAPA, were separately introduced at position 147, 150 or 151 of a short p21 peptide (141–155) known to bind sliding clamp protein PCNA. The ability of these peptides, 1a-3a and 1b-3b, to act as a turn-on fluorescent sensor for PCNA was then investigated. The 4-DMNA-containing peptides (1a-3a) displayed up to a 40-fold difference in fluorescence between a polar (Tris buffer) and a hydrophobic solvent (dioxane with 5 mM 18-crown-6), while the 4-DAPA-containing peptides (1b-3b) displayed a significantly enhanced (300-fold) increase in fluorescence from Tris buffer to dioxane with 18-crown-6. SPR analysis of the peptides against PCNA revealed that the 151-substituted peptides 3a and 3b interacted specifically with PCNA, with KD values of 921 nM and 1.28 μM, respectively. Analysis of the fluorescence of these peptides in the presence of increasing concentrations of PCNA revealed a 10-fold change in fluorescence for 3a at 2.5 equivalents of PCNA, compared to only a 3.5-fold change in fluorescence for 3b. Peptide 3a is an important lead for development of a PCNA-selective turn-on fluorescent sensor for application as a cell proliferation sensor to investigate diseases such as cancer.Aimee J.Horsfall, Theresa Chav, John B.Bruning, Andrew D.Abel

Approaches to introduce helical structure in cysteine-containing peptides with a bimane group

First published: 09 June 2021An i-i+4 or i-i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα) in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i-i+4 constrained peptide adopts a 310-helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.Aimee J. Horsfall, Daniel P. McDougal, Denis B. Scanlon, John B. Bruning, Andrew D. Abel

A bimane‐based peptide staple for combined helical induction and fluorescent imaging

The thiol-selective fluorescent imaging agent, dibromobimane, has been repurposed to crosslink cysteine- and homocysteine- containing peptides, with the resulting bimane linker acting as both a structural constraint and a fluorescent tag. Macro- cyclisation was conducted on nine short peptides containing two cysteines and/or homocysteines, both on-resin and in buffered aqueous solution, to give macrocycles ranging in size from 16 (i,i +2) to 31 (i,i + 7) atoms. The structures were defined by CD, NMR structure calculations by using Xplor-NIH, NMR secondary shift and J HαNH analyses to reveal helical structure in the i,i + 4 (1, 2), and i,i + 3 (5) constrained peptides. Cellular- uptake studies were conducted with three of the macrocycles. Subsequent confocal imaging revealed punctate fluorescence within the cytosol indicative of peptides trapped in endocytic vesicles. These studies demonstrate that dibromobimane is an effective tool for defining secondary structure within short peptides, whilst simultaneously introducing a fluorescent tag suitable for common cell-based experiments.Aimee J. Horsfall, Kylie R. Dunning, Kelly L. Keeling, Denis B. Scanlon, Kate L. Wegener, Andrew D. Abel

Protein detection enabled using functionalised silk-binding peptides on a silk-coated optical fibre

We present a new coating procedure to prepare optical fibre sensors suitable for use with protein analytes. We demonstrate this through the detection of AlexaFluor-532 tagged streptavidin by its binding to D-biotin that is functionalised onto an optical fibre, via incorporation in a silk fibroin fibre coating. The D-biotin was covalently attached to a silk-binding peptide to provide SBP–biotin, which adheres the D-biotin to the silk-coated fibre tip. These optical fibre probes were prepared by two methods. The first involves dip-coating the fibre tip into a mixture of silk fibroin and SBP–biotin, which distributes the SBP–biotin throughout the silk coating (method A). The second method uses two steps, where the fibre is first dip-coated in silk only, then SBP–biotin added in a second dip-coating step. This isolates SBP–biotin to the outer surface of the silk layer (method B). A series of fluorescence measurements revealed that only the surface bound SBP–biotin detects streptavidin with a detection limit of 15 μg mL−1. The fibre coatings are stable to repeated washing and long-term exposure to water. Formation of silk coatings on fibres using commercial aqueous silk fibroin was found to be inhibited by a lithium concentration of 200 ppm, as determined by atomic absorption spectroscopy. This was reduced to less than 20 ppm by dialysis against water, and was found to successfully form a coating on optical fibres.Patrick K. Capon, Aimee J. Horsfall, Jiawen Li, Erik P. Schartner, Asma Khalid, Malcolm S. Purdey ... et al

Designing Fluorescent Nuclear Permeable Peptidomimetics to Target Proliferating Cell Nuclear Antigen

Published 10 August 2023Human proliferating cell nuclear antigen (PCNA) is a critical mediator of DNA replication and repair, acting as a docking platform for replication proteins. Disrupting these interactions with a peptidomimetic agent presents as a promising avenue to limit proliferation of cancerous cells. Here, a p21-derived peptide was employed as a starting scaffold to design a modular peptidomimetic that interacts with PCNA and is cellular and nuclear permeable. Ultimately, a peptidomimetic was produced which met these criteria, consisting of a fluorescein tag and SV40 nuclear localization signal conjugated to the N-terminus of a p21 macrocycle derivative. Attachment of the fluorescein tag was found to directly affect cellular uptake of the peptidomimetic, with fluorescein being requisite for nuclear permeability. This work provides an important step forward in the development of PCNA targeting peptidomimetics for use as anti-cancer agents or as cancer diagnostics.Aimee J. Horsfall, Theresa Chav, Jordan L. Pederick, Zoya Kikhtyak, Bethiney C. Vandborg, Wioleta Kowalczyk, Denis B. Scanlon, Wayne D. Tilley, Theresa E. Hickey, Andrew D. Abell, and John B. Brunin

A silk‐based functionalization architecture for single fiber imaging and sensing

First published: 29 March 2021A new fiber functionalization architecture for single-fiber imaging and sensing is presented. 5(6)Carboxy-seminaphthorhodafluor-2 (a fluorescent pH sensor) is attached to a silk-binding peptide and the complex added to aqueous silk fibroin protein. These bind with a Kd of 36 µM as determined by a fluorescence polarization assay. The fiber is dip-coated into the silk and peptide mixture, and scanning electron microscopy images reveal a uniform silk coating on the fiber tip. The coating is stable to repeated washes and does not affect the imaging light emitted from the fiber, which allows concurrent optical coherence tomography (OCT) imaging and pH sensing. Oocytes are metabolically stimulated with CoCl2 to produce lactic acid, and a pH reduction of 0.04 is measured using the probe. The distance between fiber tip and oocyte is monitored by simultaneous OCT acquisitions to precisely position the probe. Lastly, OCT imaging of an ovary revealed the presence/absence of an oocyte within a follicle, an important step toward improving patient outcomes during in vitro fertilization, by limiting the number of invasive follicle punctures required. These results demonstrate the utility of this new coating to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems.Patrick K. Capon, Jiawen Li, Aimee J. Horsfall, Suliman Yagoub, Erik P. Schartner, Asma Khalid, Rodney W. Kirk, Malcolm S. Purdey, Kylie R. Dunning, Robert A. McLaughlin, and Andrew D. Abe