Biochemistry and Pharmacology - Research Publications
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ItemSialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial GrowthBozzola, T ; Scalise, M ; Larsson, CU ; Newton-Vesty, MC ; Rovegno, C ; Mitra, A ; Cramer, J ; Wahlgren, WY ; Santhakumari, PR ; Johnsson, RE ; Schwardt, O ; Ernst, B ; Friemann, R ; Dobson, RCJ ; Indiveri, C ; Schelin, J ; Nilsson, UJ ; Ellervik, U (AMER CHEMICAL SOC, 2022-06-08)Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.
ItemNo Preview AvailableField Effect Transistor-Like Control of Capillaric Flow Using Off-ValvesMeffan, RC ; Mak, D ; Menges, J ; Dolamore, F ; Fee, C ; Dobson, RCJ ; Nock, V (IEEE, 2022-01-01)
ItemNo Preview AvailableThe structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily.Love, MJ ; Coombes, D ; Ismail, S ; Billington, C ; Dobson, RCJ (Portland Press Ltd., 2022-01-28)Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.
ItemFermentation of plant-based dairy alternatives by lactic acid bacteriaHarper, AR ; Dobson, RCJ ; Morris, VK ; Moggre, G-J (WILEY, 2022-04-08)Ethical, environmental and health concerns around dairy products are driving a fast-growing industry for plant-based dairy alternatives, but undesirable flavours and textures in available products are limiting their uptake into the mainstream. The molecular processes initiated during fermentation by lactic acid bacteria in dairy products is well understood, such as proteolysis of caseins into peptides and amino acids, and the utilisation of carbohydrates to form lactic acid and exopolysaccharides. These processes are fundamental to developing the flavour and texture of fermented dairy products like cheese and yoghurt, yet how these processes work in plant-based alternatives is poorly understood. With this knowledge, bespoke fermentative processes could be engineered for specific food qualities in plant-based foods. This review will provide an overview of recent research that reveals how fermentation occurs in plant-based milk, with a focus on how differences in plant proteins and carbohydrate structure affect how they undergo the fermentation process. The practical aspects of how this knowledge has been used to develop plant-based cheeses and yoghurts is also discussed.
ItemCapillaric field effect transistors.Meffan, C ; Menges, J ; Dolamore, F ; Mak, D ; Fee, C ; Dobson, RCJ ; Nock, V (Springer Science and Business Media LLC, 2022)Controlling fluid flow in capillaric circuits is a key requirement to increase their uptake for assay applications. Capillary action off-valves provide such functionality by pushing an occluding bubble into the channel using a difference in capillary pressure. Previously, we utilized the binary switching mode of this structure to develop a powerful set of fundamental fluidic valving operations. In this work, we study the transistor-like qualities of the off-valve and provide evidence that these structures are in fact functionally complementary to electronic junction field effect transistors. In view of this, we propose the new term capillaric field effect transistor to describe these types of valves. To support this conclusion, we present a theoretical description, experimental characterization, and practical application of analog flow resistance control. In addition, we demonstrate that the valves can also be reopened. We show modulation of the flow resistance from fully open to pinch-off, determine the flow rate-trigger channel volume relationship and demonstrate that the latter can be modeled using Shockley's equation for electronic transistors. Finally, we provide a first example of how the valves can be opened and closed repeatedly.