Toll-like receptors for pathogen detection in water: challenges and benefits

The need for effective and efficient methods for pathogen detection in water is as serious as ever due to the health risk posed to human population by the consumption of pathogen-contaminated water. One of the important research streams which have been focused on by researchers for development of novel techniques for this purpose is biosensor technologies. Using different bio-recognition elements and transduction methodologies, biosensors have the potential to detect their analyte of interest in a fast and highly specific manner. Different pathogenic agents can be recognised by toll-like receptors (TLRs). The innate immune system of higher organisms employs TLRs for triggering intracellular signalling and induction of the expression of immune response genes. In this report, we explore the challenges associated with employing TLRs for pathogen detection in water samples. Although methods using TLR expressing cells also have been discussed, the focus of this review is on using TLR proteins as the bio-recognition elements in biosensors.     

Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System

Ligands of the G_i protein-coupled adenosine A₃ receptor (A₃R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A₃R. The present review summarizes information on the effect of latest-generation A₃R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A₃R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.     

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Despite the fact that transmembrane proteins represent the main therapeutic targets for decades, complete and in-depth knowledge about their biochemical and pharmacological profiling is not fully available. In this regard, target-tailored small-molecule fluorescent ligands are a viable approach to fill in the missing pieces of the puzzle. Such tools, coupled with the ability of high-precision optical techniques to image with an unprecedented resolution at a single-molecule level, helped unraveling many of the conundrums related to plasma proteins’ life-cycle and druggability. Herein, we review the recent progress made during the last two decades in fluorescent ligand design and potential applications in fluorescence microscopy of voltage-gated ion channels, ligand-gated ion channels and G-coupled protein receptors.     

Azulene‐Based Macrocyclic Receptors for Recognition and Sensing of Phosphate Anions

Herein we report the synthesis and detailed studies of the anion‐binding properties of two 20‐membered macrocyclic tetramide receptors: one symmetrical, containing two identical azulene‐based bisamide units, the other a hybrid, containing a dipicolinic bisamide unit and an azulene‐based bisamide unit. Analysis of the crystal structures of the macrocyclic receptors revealed their preference for adopting similar well‐preorganized bent‐sheet conformations, both as free receptors and in their complexes with anions. Studies of the optical properties of both receptors revealed abilities to selectively sense phosphate anions (H₂PO₄^?, HP₂O₇^3?), allowing for naked‐eye detection of the presence of these guests in DMSO. Binding studies in solution confirmed that the receptors bind strongly to a series of anions even in highly demanding media, such as mixtures of DMSO with water or with methanol. Comparison of the anion affinity of linear analogues with that of the macrocyclic receptors evidenced the importance of macrocyclic topology. Quantitative analysis revealed that the macrocyclic receptors are selective for H₂PO₄^? over other anions. The affinity to H₂PO₄^? seen for the symmetrical receptor, containing two azulene‐based subunits, is much higher than for the hybrid macrocycle containing both the azulene‐based and pyridine‐derived subunits. This highlights that the azulene‐based building block serves efficiently as both a binding site and a structure‐preorganizing motif.     

New Molecular Receptors with Cyclophosphazene Subunits: Synthesis, Reactivity, and Structure-Property Relationships

A series of hydrophopic ( 2 and 3) and new hydrophilic ( 4– 7) molecular receptors of the PNP-lariat ether with tetra-substituted cyclotriphosphazene subunits have been prepared by the complete nucleophilic substitution of chlorine atoms in the reactive PNP-crown precursor 1 with the respective sodium cation-paired oxyanions (phenoxy 2, -naphthoxy 3, and methoxytrioxyethylenoxy 4) and aliphatic amines (n-propylamine 5 aziridine 6, and pyrrolidine 7). Their structures were established by MS and ³¹P NMR spectroscopy and their metal ion complexing properties tested by a TLC method. Comparison of the complexation behaviour for ligands 1– 7 shows that the affinity for particular cations is strongly substituent-dependent and, in general, is significantly enhanced by cooperation of the side arm donor atoms (O or N) with the parent PNP-crown structure in the binding process. The remarkable affinity of some ligands for selected cations, in particular lithium, cesium, and silver ions, is interpreted in terms of structure-property relationships.     

Membrane Organization and Dynamics of the G-Protein-Coupled Serotonin<Subscript>1A</Subscript> Receptor Monitored Using Fluorescence-Based Approaches

The G-protein-coupled receptor (GPCR) superfamily represents one of the largest classes of molecules involved in signal transduction across the plasma membrane. Fluorescence-based approaches have provided valuable insights into GPCR functions such as receptor–receptor and receptor–ligand interactions, real-time assessment of signal transduction, receptor dynamics on the plasma membrane, and intracellular trafficking of receptors. This has largely been possible with the use of fluorescent probes such as the green fluorescent protein (GFP) from the jellyfish Aequoria victoria and its variants. We discuss the potential of fluorescence-based approaches in providing novel information on the membrane organization and dynamics of the G-protein-coupled serotonin_1A receptor tagged to the enhanced yellow fluorescent protein (EYFP). These authors contributed equally to the work.     

Subunit rotation models activation of serotonin 5-HT<Subscript>3AB</Subscript> receptors by agonists

The N-terminal extracellular regions of heterooligomeric 3AB-type human 5-hydroxytryptamine receptors (5-HT3ABR) were modelled based on the crystal structure of snail acetylcholine binding protein AChBP. Stepwise rotation of subunit A by 5 degrees was performed between -10 degrees and 15 degrees to mimic agonist binding and receptor activation. Anticlockwise rotation reduced the size of the binding cavity in interface AB and reorganised the network of hydrogen bonds along the interface. AB subunit dimers with different rotations were applied for docking of ligands with different efficacies: 5-HT, m-chlorophenylbiguanide, SR 57227, quinolinyl piperazine and lerisetron derivatives. All ligands were docked into the dimer with -10 degrees rotation representing ligand-free, open binding cavities similarly, without pharmacological discrimination. Their ammonium ions were in hydrogen bonding distance to the backbone carbonyl of W183. Anticlockwise rotation and contraction of the binding cavity led to distinctive docking interactions of agonists with E129 and cation-pi interactions of their ammonium ions. Side chains of several further amino acids participating in docking (Y143, Y153, Y234 and E236) are in agreement with the effects of point mutations in the binding loops. Our model postulates that 5-HT binds to W183 in a hydrophobic cleft as well as to E236 in a hydrophilic vestibule. Then it elicits anticlockwise rotation to draw in loop C via pi-cation-pi interactions of its ammonium ion with W183 and Y234. Finally, closure of the binding cavity might end in rebinding of 5-HT to E129 in the hydrophilic vestibule.