10 research outputs found
Recommended from our members
Fluorescence spectroscopy analysis of the bacteria-mineral interface: adsorption of lipopolysaccharides to silica and alumina
YesWe present here a quantification of the sorption process and molecular conformation involved in the attachment of bacterial cell wall lipopolysaccharides (LPSs), extracted from Escherichia coli, to silica (SiO2) and alumina (Al2O3) particles. We propose that interfacial forces govern the physicochemical interactions of the bacterial cell wall with minerals in the natural environment, and the molecular conformation of LPS cell wall components depends on both the local charge at the point of binding and hydrogen bonding potential. This has an effect on bacterial adaptation to the host environment through adhesion, growth, function, and ability to form biofilms. Photophysical techniques were used to investigate adsorption of fluorescently labeled LPS onto mineral surfaces as model systems for bacterial attachment. Adsorption of macromolecules in dilute solutions was studied as a function of pH and ionic strength in the presence of alumina and silica via fluorescence, potentiometric, and mass spectrometry techniques. The effect of silica and alumina particles on bacterial growth as a function of pH was also investigated using spectrophotometry. The alumina and silica particles were used to mimic active sites on the surface of clay and soil particles, which serve as a point of attachment of bacteria in natural systems. It was found that LPS had a high adsorption affinity for Al2O3 while adsorbing weakly to SiO2 surfaces. Strong adsorption was observed at low pH for both minerals and varied with both pH and mineral concentration, likely in part due to conformational rearrangement of the LPS macromolecules. Bacterial growth was also enhanced in the presence of the particles at low pH values. This demonstrates that at a molecular level, bacterial cell wall components are able to adapt their conformation, depending on the solution pH, in order to maximize attachment to substrates and guarantee community survival.The authors thank the Libyan Ministry of Education for financial support during the experimental study. We thank the EPSRC funded consortium “Hard-soft matter interfaces: from understanding to engineering” (EP/I001514/1) for financial support. Emily Caseley, who assisted in the preparation and characterization of AmNS-LPS particles as an MRC Confidence in Concept funded postdoctoral researcher at the University of Bradford, (MC_PC_16038)
Neurodegenerative Disease and the NLRP3 Inflammasome.
The prevalence of neurodegenerative disease has increased significantly in recent years, and with a rapidly aging global population, this trend is expected to continue. These diseases are characterised by a progressive neuronal loss in the brain or peripheral nervous system, and generally involve protein aggregation, as well as metabolic abnormalities and immune dysregulation. Although the vast majority of neurodegeneration is idiopathic, there are many known genetic and environmental triggers. In the past decade, research exploring low-grade systemic inflammation and its impact on the development and progression of neurodegenerative disease has increased. A particular research focus has been whether systemic inflammation arises only as a secondary effect of disease or is also a cause of pathology. The inflammasomes, and more specifically the NLRP3 inflammasome, a crucial component of the innate immune system, is usually activated in response to infection or tissue damage. Dysregulation of the NLRP3 inflammasome has been implicated in the progression of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. This review aims to summarise current literature on the role of the NLRP3 inflammasome in the pathogenesis of neurodegenerative diseases, and recent work investigating NLRP3 inflammasome inhibition as a potential future therapy
Escape from adamantane: scaffold optimization of novel P2X7 antagonists featuring complex polycycles
The adamantane scaffold, despite being widely used in medicinal chemistry, is not devoid of problems. In recent years we have developed new polycyclic scaffolds as surrogates of the adamantane group with encouraging results in multiple targets. As an adamantane scaffold is a common structural feature in several P2X7 receptor antagonists, herein we report the synthesis and pharmacological evaluation of multiple replacement options of adamantane that maintain a good activity profile. Molecular modeling studies support the binding of the compounds to a site close to the central pore, rather than to the ATP-binding site and shed light on the structural requirements for novel P2X7 antagonists
Docking of competitive inhibitors to the P2X7 receptor family reveals key differences responsible for changes in response between rat and human
The P2X7 receptor is a calcium permeable cationic channel activated by extracellular ATP, playing a role in chronic pain, osteoporosis and arthritis. A number of potential lead compounds are inactive against the rat isoform, despite good activity against the human homologue, making animal model studies problematic. Here we have produced P2X7 models and docked three structurally distinct inhibitors using in silico approaches and show they have a similar mode of binding in which Phe95 plays a key role by forming pi-stacking interactions. Importantly this residue is replaced by Leu in the rat P2X7 receptor resulting in a significantly reduced binding affinity. This work provides new insights into binding of P2X7 inhibitors and shows the structural difference in human and rat P2X7 receptors which results in a difference in affinity. Such information is useful both for the rational design of inhibitors based on these scaffolds and also the way in which these compounds are tested in animal models
Conformational changes during human P2X7 receptor activation examined by structural modelling and cysteine-based cross-linking studies
The P2X7 receptor (P2X7R) is important in mediating a range of physiological functions and pathologies associated with tissue damage and inflammation and represents an attractive therapeutic target. However, in terms of their structure-function relationships, the mammalian P2X7Rs remain poorly characterised compared to some of their other P2XR counterparts. In this study, combining cysteine-based cross-linking and whole-cell patch-clamp recording, we examined six pairs of residues (A44/I331, D48/I331, I58/F311, S60/L320, I75/P177 and K81/V304) located in different parts of the extracellular and transmembrane domains of the human P2X7R. These residues are predicted to undergo substantial movement during the transition of the receptor ion channel from the closed to the open state, predictions which are made based on structural homology models generated from the crystal structures of the zebrafish P2X4R. Our results provide evidence that among the six pairs of cysteine mutants, D48C/I133C and K81C/V304C formed disulphide bonds that impaired the channel gating to support the notion that such conformational changes, particularly those in the outer ends of the transmembrane domains, are critical for human P2X7R activation
Non-Synonymous Single Nucleotide Polymorphisms in the P2X Receptor Genes: Association with Diseases, Impact on Receptor Functions and Potential Use as Diagnosis Biomarkers
P2X receptors are Ca2+-permeable cationic channels in the cell membranes, where they play an important role in mediating a diversity of physiological and pathophysiological functions of extracellular ATP. Mammalian cells express seven P2X receptor genes. Single nucleotide polymorphisms (SNPs) are widespread in the P2RX genes encoding the human P2X receptors, particularly the human P2X7 receptor. This article will provide an overview of the non-synonymous SNPs (NS-SNPs) that have been associated with or implicated in altering the susceptibility to pathologies or disease conditions, and discuss the consequences of the mutations resulting from such NS-SNPs on the receptor functions. Disease-associated NS-SNPs in the P2RX genes have been valuable in understanding the disease etiology and the receptor function, and are promising as biomarkers to be used for the diagnosis and development of stratified therapeutics
Structural Basis for Ligand-Receptor Interactions at the P2X7 Receptor
P2X7 receptors (P2X7Rs) belong to the P2X receptor family of ligand-gated ion channels activated by extracellular ATP. The human P2X7R (hP2X7R) is implicated in numerous debilitating disease conditions and thus represents a promising therapeutic target. However, P2X7R structure-function relationships remain less well understood. The study presented in this thesis used electrophysiology in conjunction with structural modelling, molecular docking and site-directed mutagenesis to better understand the structural basis for ligand-receptor interactions at P2X7Rs and used such structural information to identify novel hP2X7R antagonists.
Initially, P2X7R homology models were produced based on the crystal structures of the zebrafish P2X4R (zfP2X4R) in closed and ATP-bound states and validated through docking and biochemical approaches. First of all, molecular docking showed that ATP binds to the zfP2X4R and hP2X7R in a strikingly similar configuration to the crystal structure. Secondly, docking of the antagonists AZ11645373, KN62 and SB203580 revealed a specific interaction with Phe95 in the hP2X7R that is absent in the rat P2X7R (rP2X7R), providing structural insight into their preferential hP2X7R antagonism. Thirdly, replacing Asp48 and Ile331 with cysteine resulted in disulfide bonding that impaired hP2X7R-mediated currents, which was reversibly restored by dithiothreitol. These results are consistent with the transmembrane domains moving substantially apart, as predicted by the closed and open state models. Overall, these experiments show that homology models can yield meaningful structural information in terms of P2X7R interactions with ATP, antagonists and receptor activation.
The second part of the study searched for P2X7R antagonists using a structure-based approach. Virtual screening of ~100,000 compounds in the ZINC library against the ATP-binding pocket in the hP2X7R model identified C23, C40 and C60 as structurally novel antagonists of the hP2X7R but not the rP2X7R. These compounds inhibited the agonist-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) with IC50 values in the micromolar range. C23 and C40 also inhibited agonist-induced currents with similar potency, but C60 did not. All three compounds suppressed large pore formation with micromolar potency. While C23 inhibited agonist-induced [Ca2+]i increase mediated by the hP2X4R and rP2X3R, C40 and C60 were more selective towards the hP2X7R. In conclusion, these results show C23, C40 and C60 as novel hP2X7R antagonists. Such structure-based approaches should aid novel P2XR antagonist identification.
Finally, the models were used in combination with site-directed mutagenesis to investigate residues influencing hP2X7R-agonist interactions. The first set of experiments examined four residues implicated in interactions with ATP, and only the mutation of Tyr288 to various residues significantly affected agonist sensitivity. This Tyr288 mutation abolished receptor function, which was mainly due to impairment in protein surface expression as shown by immunofluorescent imaging. The second set of experiments examined several residues for their contribution in the difference in functional expression and agonist sensitivity. Substitution of Val87 in the hP2X7R for Ile in the rP2X7R increased the maximal agonist-induced currents.
Overall, the present study provides structural insights into ligand-receptor interactions at the P2X7Rs. It demonstrates that structure-based approaches are feasible in identifying novel antagonists, and this has wider implications for the P2XR family and membrane proteins as a whole
Escape from adamantane: scaffold optimization of novel P2X7 antagonists featuring complex polycycles
The adamantane scaffold, despite being widely used in medicinal chemistry, is not devoid of problems. In recent years we have developed new polycyclic scaffolds as surrogates of the adamantane group with encouraging results in multiple targets. As an adamantane scaffold is a common structural feature in several P2X7 receptor antagonists, herein we report the synthesis and pharmacological evaluation of multiple replacement options of adamantane that maintain a good activity profile. Molecular modeling studies support the binding of the compounds to a site close to the central pore, rather than to the ATP-binding site and shed light on the structural requirements for novel P2X7 antagonists
Escape from adamantane: scaffold optimization of novel P2X7 antagonists featuring complex polycycles
The adamantane scaffold, despite being widely used in medicinal chemistry, is not devoid of problems. In recent years we have developed new polycyclic scaffolds as surrogates of the adamantane group with encouraging results in multiple targets. As an adamantane scaffold is a common structural feature in several P2X7 receptor antagonists, herein we report the synthesis and pharmacological evaluation of multiple replacement options of adamantane that maintain a good activity profile. Molecular modeling studies support the binding of the compounds to a site close to the central pore, rather than to the ATP-binding site and shed light on the structural requirements for novel P2X7 antagonists