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Journal articleCraven G, Affron D, Raymond P, et al., 2019,
Vinyl sulfonamide synthesis for irreversible tethering via a novel α-selenoether protection strategy
, MedChemComm, Vol: 10, Pages: 158-163, ISSN: 2040-2503Vinyl sulfonamides are valuable electrophiles for targeted protein modification and inhibition. We describe a novel approach to the synthesis of terminal vinyl sulfonamides which uses mild oxidative conditions to induce elimination of an α-selenoether masking group. The method complements traditional synthetic approaches and typically yields vinyl sulfonamides in high purity after aqueous work-up without requiring column chromatography of the final electrophilic product. The methodology is applied to the synthesis of covalent fragments for use in irreversible protein tethering and crucially enables the attachment of diverse fragments to the vinyl sulfonamide warhead via a chemical linker. Using thymidylate synthase as a model system, ethylene glycol is identified as an effective linker for irreversible protein tethering.
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Journal articleMenny A, Serna M, Boyd C, et al., 2018,
CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers
, Nature Communications, Vol: 9, ISSN: 2041-1723The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.
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Journal articleMiller RM, Cabral J, Robles E, et al., 2018,
Crystallisation of sodium dodecyl sulfate–water micellar solutions with structurally similar additives: counterion variation
, CrystEngComm, Vol: 20, Pages: 6834-6843, ISSN: 1466-8033The effects of a series of structurally similar sodium dodecyl sulfate (SDS) additives on the crystallisation of SDS–water micellar solutions were investigated using a combination of differential scanning calorimetry, dynamic light scattering, optical microscopy and inductively coupled plasma optical emission spectroscopy. Seven different counterions were chosen from groups 1 and 2 of the periodic table to replace the sodium on SDS: LDS, (SDS), KDS, RbDS, CsDS, Mg(DS)2, Ca(DS)2 and Sr(DS)2. Two representative temperature profileswere employed – linear cooling ramps at rate of 0.5 °C min−1 to determine near-equilibrium kinetics and transitions and isothermal holds at 6 °C to elucidate morphological changes. Crystallisation of the reference solution 20% SDS–H2O with 0.25, 1.0 and 2.5% additive was generally promoted or inhibited even at the lowest concentrations. Melting points however remained largely unchanged, suggesting that the additives predominantly had a kinetic rather than thermodynamic effect. ICP-OES measurements for the solutions containing 1% additive indicated that most of the additives were integrated into the SDS crystals which was reflected by morphological changes, including the formation of hexagonal and oval shaped crystals. Our results both quantify and provide a morphological insight into the effect of a series of additives on the crystallisation of micellar SDS solutions, which can readily form due to preferential Na exchange.
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Journal articleBarlow N, Kusumaatmaja H, Salehi-Reyhani A, et al., 2018,
Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers
, Journal of the Royal Society Interface, Vol: 15, ISSN: 1742-5662For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.
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Journal articleGirvan P, Teng X, Brooks NJ, et al., 2018,
Redox Kinetics of the Amyloid-β-Cu Complex and Its Biological Implications
, BIOCHEMISTRY, Vol: 57, Pages: 6228-6233, ISSN: 0006-2960- Author Web Link
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Journal articleChavas TEJ, Fuchter MJ, DiMaggio PA, 2018,
Unbiased mass spectrometry elucidation of the targets and mechanisms of activity-based probes: A case study involving sulfonyl fluorides
, ACS Chemical Biology, Vol: 13, Pages: 2897-2907, ISSN: 1554-8929The elucidation of protein/drug interactions remains a major challenge in drug discovery. Liquid chromatography–tandem mass spectrometry has emerged as a tremendously powerful technology for this endeavor, but its full potential has yet to be realized owing in part to unresolved challenges in data analysis. Herein, we demonstrate how tandem mass spectrometry can comprehensively map small molecule/peptide adducts when combined with unconstrained sequencing. Using a published sulfonyl fluoride activity-based probe as a model system, this method enabled the discovery of several unreported sites of interaction with its target proteins. Crucially, this probe was found to undergo quantitative displacement and hydrolysis from the target protein’s active site. Isotopic labeling experiments provided a mechanistic rationale for the observed hydrolysis that involves neighboring-group participation. A chemical biology tagging strategy that leverages the probe’s observed lability was developed and shown to be compatible with the original small molecule inhibitor in discovery profiling experiments.
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Journal articleWang Z, Grosskurth SE, Cheung T, et al., 2018,
Pharmacological inhibition of PARP6 triggers multipolar spindle formation and demonstrates therapeutic effects in breast cancer
, Cancer Research, Vol: 78, Pages: 6691-6702, ISSN: 1538-7445PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity.
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Journal articleTrantidou T, Dekker L, Polizzi K, et al., 2018,
Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors
, Interface Focus, Vol: 8, ISSN: 2042-8901The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.
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Journal articleZhang H, Salazar JD, Yaliraki SN, 2018,
Proteins across scales through graph partitioning: application to the major peanut allergen Ara h 1
, Journal of Complex Networks, Vol: 6, Pages: 679-692, ISSN: 2051-1310The analysis of community structure in complex networks has been given much attention recently, as it is hoped that the communities at various scales can affect or explain the global behaviour of the system. A plethora of community detection algorithms have been proposed, insightful yet often restricted by certain inherent resolutions. Proteins are multi-scale biomolecular machines with coupled structural organization across scales, which is linked to their function. To reveal this organization, we applied a recently developed multi-resolution method, Markov Stability, which is based on atomistic graph partitioning, along with theoretical mutagenesis that further allows for hot spot identification using Gaussian process regression. The methodology finds partitions of a graph without imposing a particular scale a priori and analyses the network in a computationally efficient way. Here, we show an application on peanut allergenicity, which despite extensive experimental studies that focus on epitopes, groups of atoms associated with allergenic reactions, remains poorly understood. We compare our results against available experiment data, and we further predict distal regulatory sites that may significantly alter protein dynamics.
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Journal articleChatzimichail S, Supramaniam P, Ces O, et al., 2018,
Micropatterning of planar metal electrodes by vacuum filling microfluidic channel geometries
, Scientific Reports, Vol: 8, ISSN: 2045-2322We present a simple, facile method to micropattern planar metal electrodes defined by the geometry of a microfluidic channel network template. By introducing aqueous solutions of metal into reversibly adhered PDMS devices by desiccation instead of flow, we are able to produce difficult to pattern “dead end” or discontinuous features with ease. We characterize electrodes fabricated using this method and perform electrical lysis of mammalian cancer cells and demonstrate their use as part of an antibody capture assay for GFP. Cell lysis in microwell arrays is achieved using the electrodes and the protein released is detected using an antibody microarray. We show how the template channels used as part of the workflow for patterning the electrodes may be produced using photolithography-free methods, such as laser micromachining and PDMS master moulding, and demonstrate how the use of an immiscible phase may be employed to create electrode spacings on the order of 25 – 50 μm, that overcome the current resolution limits of such methods. This work demonstrates how the rapid prototyping of electrodes for use in total analysis systems can be achieved on the bench with little or no need for centralized facilities.
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