Publications by authors named "W Bruce Turnbull"

107 Publications

Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection and Nanoparticle Assembly.

J Am Chem Soc 2020 10 29;142(42):18022-18034. Epub 2020 Sep 29.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.
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http://dx.doi.org/10.1021/jacs.0c06793DOI Listing
October 2020

Correction: Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation.

Org Biomol Chem 2020 Aug;18(30):5982

Department of Chemistry, University of York, Heslington, YO10 5DD, York, UK.

Correction for 'Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation' by Robin L. Brabham et al., Org. Biomol. Chem., 2020, 18, 4000-4003, DOI: 10.1039/D0OB00972E.
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http://dx.doi.org/10.1039/d0ob90100hDOI Listing
August 2020

Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation.

Org Biomol Chem 2020 Jun 19;18(21):4000-4003. Epub 2020 May 19.

Department of Chemistry, University of York, Heslington, YO10 5DD, York, UK.

The α-oxo aldehyde is a highly reactive aldehyde for which many protein bioconjugation strategies exist. Here, we explore the genetic incorporation of a threonine-lysine dipeptide into proteins, harbouring a "masked"α-oxo aldehyde that is rapidly unveiled in four minutes. The reactive aldehyde could undergo site-specific protein modification by SPANC ligation.
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http://dx.doi.org/10.1039/d0ob00972eDOI Listing
June 2020

Chemoenzymatic synthesis of 3-deoxy-3-fluoro-l-fucose and its enzymatic incorporation into glycoconjugates.

Chem Commun (Camb) 2020 Jun;56(47):6408-6411

School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK.

The first synthesis of 3-deoxy-3-fluoro-l-fucose is presented, which employs a d- to l-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-l-fucitol. Enzymatic activation (FKP) and glycosylation using an α-1,2 and an α-1,3 fucosyltransferase to obtain two fluorinated trisaccharides demonstrates its potential as a novel versatile chemical probe in glycobiology.
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http://dx.doi.org/10.1039/d0cc02209hDOI Listing
June 2020

A versatile cholera toxin conjugate for neuronal targeting and tracing.

Chem Commun (Camb) 2020 Jun 1;56(45):6098-6101. Epub 2020 May 1.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by 'click' chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using 'click' chemistry.
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http://dx.doi.org/10.1039/d0cc01085eDOI Listing
June 2020

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

Org Biomol Chem 2020 04;18(16):3142-3148

Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.
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http://dx.doi.org/10.1039/d0ob00407cDOI Listing
April 2020

A study of Tc/Re-tricarbonyl complexes of 4-amino-1,8-naphthalimides.

Dalton Trans 2019 Oct 6;48(37):14077-14084. Epub 2019 Sep 6.

Department of Chemistry, University of Western Ontario, London, Ontario N6A 3K7, Canada. and London Regional Cancer Program, London, Ontario N6A 4L6, Canada and Lawson Health Research Institute, London, Ontario N6C 2R5, Canada and Departments of Oncology, and Medical Imaging, University of Western Ontario, London N6A 3K7, Canada.

Three 4-amino-1,8-naphthalimide analogues were synthesized, consisting of the tridentate chelators di-2-picolylamine, (pyridin-2-ylmethyl)glycinate, and iminodiacetate conjugated to the naphthalimide scaffold. Coordination with fac-Tc/Re(CO) resulted in metal complexes with overall charges of -1, 0, or +1. Upon coordination of Re(i), the initial naphthalimide-based fluorescence is largely maintained for both negative and neutral complexes compared to their free ligand forms, while an increase in fluorescence quantum yield was observed for the positively charged complex. OVCAR-8 ovarian cancer cells were stained with each of the complexes, demonstrating that the positive complex is more lipophilic and cell membrane permeable than the neutral and negative complexes. Each of the three technetium-99m labelled naphthalimide complexes were successfully produced from fac-[Tc(CO)(HO)] in 15 minutes at 70 °C and isolated in radiochemical yields ranging from 60-95% with radiochemical purities greater than 95%. These fluorescent metal complexes of various charges can be used to tune pharmacokinetic and cellular uptake properties of Tc/Re-naphthalimide-bioconjugates, while still maintaining desirable fluorescence properties.
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http://dx.doi.org/10.1039/c9dt01752fDOI Listing
October 2019

Comprehensive in vitro characterization of PD-L1 small molecule inhibitors.

Sci Rep 2019 08 27;9(1):12392. Epub 2019 Aug 27.

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.
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http://dx.doi.org/10.1038/s41598-019-48826-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712002PMC
August 2019

A 'catch-and-release' receptor for the cholera toxin.

Faraday Discuss 2019 10;219(0):112-127

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK.

Stimuli-responsive receptors for the recognition unit of the cholera toxin (CTB) have been prepared by attaching multiple copies of its natural carbohydrate ligand, the GM1 oligosaccharide, to a thermoresponsive polymer scaffold. Below their lower critical solution temperature (LCST), polymers complex CTB with nanomolar affinity. When heated above their LCST, polymers undergo a reversible coil to globule transition which renders a proportion of the carbohydrate recognition motifs inaccessible to CTB. This thermally-modulated decrease in the avidity of the material for the protein has been used to reversibly capture CTB from solution, enabling its convenient isolation from a complex mixture.
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http://dx.doi.org/10.1039/c9fd00017hDOI Listing
October 2019

Enzymatic synthesis of N-acetyllactosamine from lactose enabled by recombinant β1,4-galactosyltransferases.

Org Biomol Chem 2019 06;17(24):5920-5924

Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester, 131 Princess Street, M1 7DN, Manchester, UK.

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial β1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-β-LacNAc from lactose using β1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.
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http://dx.doi.org/10.1039/c9ob01089kDOI Listing
June 2019

Directed Assembly of Homopentameric Cholera Toxin B-Subunit Proteins into Higher-Order Structures Using Coiled-Coil Appendages.

J Am Chem Soc 2019 04 21;141(13):5211-5219. Epub 2019 Mar 21.

Astbury Centre for Structural Molecular Biology , University of Leeds , Leeds LS2 9JT , United Kingdom.

The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein-protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for "designability", limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight "native-state" forced protein-protein interfaces, which may prove useful as starting conformations for future computational design.
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http://dx.doi.org/10.1021/jacs.8b11480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449800PMC
April 2019

A dual modality Tc/Re(i)-labelled T140 analogue for imaging of CXCR4 expression.

Org Biomol Chem 2019 01;17(3):598-608

Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B7, Canada.

The C-X-C chemokine receptor 4 (CXCR4) has been shown to be overexpressed in at least 23 types of cancer, including prostate cancer which has been shown to have a significant distinction of expression rates between cancerous compared to healthy or benign tissue. In an attempt to exploit the difference in expression, we have synthesized a derivative of T140, a peptide antagonist for CXCR4, containing a fluorescent 4-amino-1,8-naphthalimide appended with a di-(2-picolyl)amine binding unit to chelate rhenium or technetium-99m for fluorescence or SPECT imaging. The rhenium-coordinated variant was shown to have similar binding affinity for the receptor as T140 and showed specific uptake by fluorescence microscopy in CXCR4 expressing cells. The peptide was radiolabelled with technetium-99m in decay corrected radiochemical yields ranging from 60-85%, radiochemical purities >95%, and molar activities of 36-44 GBq μmol-1. The technetium-99m labelled peptide showed two-fold higher uptake in U87 cells expressing CXCR4 compared to non-transfected cells. Ex vivo biodistribution studies were performed using the technetium-99m labelled peptide in NOD/SCID mice bearing tumors derived from U87 cells with CXCR4. Tumor uptake of 0.51 ± 0.09% ID g-1 was observed two-hours post-injection. Our novel T140 derivative is suitable for imaging of CXCR4 expression by confocal microscopy. Further structural modifications to the peptide or metal complex may result in improved biodistribution for use in SPECT imaging of CXCR4 expressing tumors.
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http://dx.doi.org/10.1039/c8ob01947aDOI Listing
January 2019

Amino-Substituted 2,2'-Bipyridine Ligands as Fluorescent Indicators for Zn and Applications for Fluorescence Imaging of Prostate Cells.

Chemistry 2018 Sep 5;24(54):14539-14546. Epub 2018 Sep 5.

Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 5B7, Canada.

Zn concentrations in malignant prostate tissues are much lower than in benign or healthy, suggesting that Zn levels are a potential biomarker for prostate cancer (PCa). Five 2,2'-bipyridine ligands were synthesized containing amino substituents with varying electron-donating ability for investigation as fluorescent Zn indicators. The excited state characteristics of the ligands were explored by UV/Vis and fluorescence spectroscopy. 3,3'-Diamino-2,2'-bipyridine (1) was previously shown to be weakly fluorescent as a result of π→π* transitions. The other four ligands have properties consistent with an n→π* intraligand charge transfer excited state. Strongly donating amino and aminophenyl (2 and 4) substituents gave low quantum yields, while weaker donating benzimidazole substituents (6 and 7) gave high quantum yields. Absorption and fluorescence wavelengths underwent bathochromic shifts upon Zn binding in a majority of cases. Quantum yields drastically increased upon Zn binding for 1 and 2, but decreased for 4, 6, and 7. Compounds 6 and 7 were incubated with PC-3, DU 145 and BPH-1 cells to determine their Zn sensing abilities in a biological system. Weak fluorescence was observed in BPH-1 cells and subsequent incubation with Zn caused fluorescence intensity to increase. No fluorescence was observed in PCa cell lines. Further investigation of these ligands may allow for quantitative determination of Zn concentrations in ex vivo tissue samples.
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http://dx.doi.org/10.1002/chem.201803051DOI Listing
September 2018

Carbohydrate inhibitors of cholera toxin.

Beilstein J Org Chem 2018 21;14:484-498. Epub 2018 Feb 21.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT, UK.

Cholera is a diarrheal disease caused by a protein toxin released by in the host's intestine. The toxin enters intestinal epithelial cells after binding to specific carbohydrates on the cell surface. Over recent years, considerable effort has been invested in developing inhibitors of toxin adhesion that mimic the carbohydrate ligand, with particular emphasis on exploiting the multivalency of the toxin to enhance activity. In this review we introduce the structural features of the toxin that have guided the design of diverse inhibitors and summarise recent developments in the field.
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http://dx.doi.org/10.3762/bjoc.14.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827775PMC
February 2018

Molecular Recognition-Mediated Transformation of Single-Chain Polymer Nanoparticles into Crosslinked Polymer Films.

Angew Chem Int Ed Engl 2017 10 8;56(42):12913-12918. Epub 2017 Sep 8.

Chemical Nanoscience Laboratory, School of Chemistry, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK.

We describe single-chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition-mediated crosslinking process. The SCNPs utilise molecular recognition with surface-immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra- to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to "wrap" surfaces displaying molecular recognition motifs-which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs-within a layer of polymer film.
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http://dx.doi.org/10.1002/anie.201706379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656938PMC
October 2017

Dissecting Multivalent Lectin-Carbohydrate Recognition Using Polyvalent Multifunctional Glycan-Quantum Dots.

J Am Chem Soc 2017 08 17;139(34):11833-11844. Epub 2017 Aug 17.

Infection Biology Unit, German Primate Center , Kellnerweg 4, Gottingen 37077, Germany.

Multivalent protein-carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein-glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN's higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent K = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein-ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.
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http://dx.doi.org/10.1021/jacs.7b05104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579584PMC
August 2017

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

Angew Chem Weinheim Bergstr Ger 2016 Apr 15;128(15):4816-4820. Epub 2016 Mar 15.

School of Chemistry and Astbury Centre for Structural Molecular Biology University of Leeds Leeds LS2 9JT UK.

A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions.
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http://dx.doi.org/10.1002/ange.201600593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979676PMC
April 2016

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

Angew Chem Int Ed Engl 2016 Apr 16;55(15):4738-42. Epub 2016 Mar 16.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions.
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http://dx.doi.org/10.1002/anie.201600593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979658PMC
April 2016

Molecular imaging probes derived from natural peptides.

Nat Prod Rep 2016 06;33(6):761-800

Department of Chemistry, The University of Western Ontario, London, Canada. and Departments of Oncology and Medical Imaging, The University of Western Ontario, London, Canada and London Regional Cancer Program, Lawson Health Research Institute, London, Canada.

Covering: up to the end of 2015.Peptides are naturally occurring compounds that play an important role in all living systems and are responsible for a range of essential functions. Peptide receptors have been implicated in disease states such as oncology, metabolic disorders and cardiovascular disease. Therefore, natural peptides have been exploited as diagnostic and therapeutic agents due to the unique target specificity for their endogenous receptors. This review discusses a variety of natural peptides highlighting their discovery, endogenous receptors, as well as their derivatization to create molecular imaging agents, with an emphasis on the design of radiolabelled peptides. This review also highlights methods for discovering new and novel peptides when knowledge of specific targets and endogenous ligands are not available.
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http://dx.doi.org/10.1039/c5np00083aDOI Listing
June 2016

Confirmation of a Protein-Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase-Mediated Labelling.

Chembiochem 2016 Apr 1;17(8):753-8. Epub 2016 Mar 1.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

High-throughput studies have been widely used to identify protein-protein interactions; however, few of these candidate interactions have been confirmed in vitro. We have used a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observed no interaction between the next enzyme in the pathway, pantothenate synthetase (PS), and aspartate decarboxylase, but did observe an interaction between PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling an adventitiously formed glycine on the protein N-terminal affinity purification tag by using Sortase. Subsequent formation of the protein-protein complex led to apparent restriction of the dynamics of this tag, thus suggesting that this approach could be generally applied to a subset of other protein-protein interaction complexes.
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http://dx.doi.org/10.1002/cbic.201500547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963676PMC
April 2016

Mechanistic Investigations into the Application of Sulfoxides in Carbohydrate Synthesis.

Chemistry 2016 Mar 7;22(12):3916-28. Epub 2016 Jan 7.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK.

The utility of sulfoxides in a diverse range of transformations in the field of carbohydrate chemistry has seen rapid growth since the first introduction of a sulfoxide as a glycosyl donor in 1989. Sulfoxides have since developed into more than just anomeric leaving groups, and today have multiple roles in glycosylation reactions. These include as activators for thioglycosides, hemiacetals, and glycals, and as precursors to glycosyl triflates, which are essential for stereoselective β-mannoside synthesis, and bicyclic sulfonium ions that facilitate the stereoselective synthesis of α-glycosides. In this review we highlight the mechanistic investigations undertaken in this area, often outlining strategies employed to differentiate between multiple proposed reaction pathways, and how the conclusions of these investigations have and continue to inform upon the development of more efficient transformations in sulfoxide-based carbohydrate synthesis.
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http://dx.doi.org/10.1002/chem.201503504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794778PMC
March 2016

Tetra- versus Pentavalent Inhibitors of Cholera Toxin.

ChemistryOpen 2015 Aug 21;4(4):471-7. Epub 2015 Mar 21.

Department of Medicinal Chemistry and Chemical Biology, Utrecht University P.O. Box 80082, 3508 TB, Utrecht, The Netherlands.

The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface. Such simultaneous binding arising from the toxins multivalency is believed to enhance its affinity. Thus, blocking the initial attachment of the toxin to the cell surface using inhibitors with GM1 subunits has the potential to stop the disease. Previously we showed that tetravalent GM1 molecules were sub-nanomolar inhibitors of CTB5. In this study, we synthesized a pentavalent version and compared the binding and potency of penta- and tetravalent cholera toxin inhibitors, based on the same scaffold, for the first time. The pentavalent geometry did not yield major benefits over the tetravalent species, but it was still a strong inhibitor, and no major steric clashes occurred when binding the toxin. Thus, systems which can adopt more geometries, such as those described here, can be equally potent, and this may possibly be due to their ability to form higher-order structures or simply due to more statistical options for binding.
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http://dx.doi.org/10.1002/open.201500006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603408PMC
August 2015

Templating carbohydrate-functionalised polymer-scaffolded dynamic combinatorial libraries with lectins.

Org Biomol Chem 2015 Mar;13(9):2756-61

Chemical Nanoscience Laboratory, School of Chemistry, Newcastle University, Newcastle-upon-Tyne, UK.

A conceptually new approach to the design of macromolecular receptors for lectins is outlined. Carbohydrate-functionalised Polymer-Scaffolded Dynamic Combinatorial Libraries (PS-DCLs) have been prepared in aqueous solution by the reversible conjugation of carbohydrates possessing acylhydrazide functionalities in their aglycone on to an aldehyde-functionalised polymer scaffold. PS-DCLs have been shown to undergo compositional change in response to the addition of lectin templates, with polymer scaffolds preferentially incorporating carbohydrate units which recognise the lectin added. This compositional change has been shown to generate polymers of significantly enhanced affinity for the lectin added, with enhancements in free energy of binding in the range of 5.2-8.8 kJ mol(-1) observed. Experiments indicate that these enhancements are not only as a consequence of increased display of the preferred carbohydrate upon the polymer scaffold, but that templation also reorganises key residues into strategic positions in order to interact more strongly with the target.
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http://dx.doi.org/10.1039/c4ob02587cDOI Listing
March 2015

A protein-based pentavalent inhibitor of the cholera toxin B-subunit.

Angew Chem Int Ed Engl 2014 Aug 2;53(32):8323-7. Epub 2014 Jul 2.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT (UK).

Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pM for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.
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http://dx.doi.org/10.1002/anie.201404397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4499251PMC
August 2014

Depsipeptide substrates for sortase-mediated N-terminal protein ligation.

Nat Protoc 2014 Feb 9;9(2):253-62. Epub 2014 Jan 9.

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.

Technologies that allow the efficient chemical modification of proteins under mild conditions are widely sought after. Sortase-mediated peptide ligation provides a strategy for modifying the N or C terminus of proteins. This protocol describes the use of depsipeptide substrates (containing an ester linkage) with sortase A (SrtA) to completely modify proteins carrying a single N-terminal glycine residue under mild conditions in 4-6 h. The SrtA-mediated ligation reaction is reversible, so most labeling protocols that use this enzyme require a large excess of both substrate and sortase to produce high yields of ligation product. In contrast, switching to depsipeptide substrates effectively renders the reaction irreversible, allowing complete labeling of proteins with a small excess of substrate and catalytic quantities of sortase. Herein we describe the synthesis of depsipeptide substrates that contain an ester linkage between a threonine and glycolic acid residue and an N-terminal FITC fluorophore appended via a thiourea linkage. The synthesis of the depsipeptide substrate typically takes 2-3 d.
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http://dx.doi.org/10.1038/nprot.2014.003DOI Listing
February 2014