Preparation and preliminary evaluation of a tritium-labeled allosteric P2X4 receptor antagonist.

P2X4 receptors are ATP-gated cation channels that were proposed as novel drug targets due to their role in inflammation and neuropathic pain. Only few potent and selective P2X4 receptor antagonists have been described to date. Labeled tool compounds suitable for P2X4 receptor binding studies are lacking. Here, we present a novel allosteric P2X4 receptor antagonist possessing high potency in the low nanomolar range. We describe its tritium-labeling resulting in the P2X4-selective radiotracer [3H]PSB-OR-2020 with high specific activity (45 Ci/mmol; 1.67 TBq/mmol). A radioligand binding assay was developed using human embryonic kidney (HEK293) cell membranes recombinantly expressing the human P2X4 receptor. Competition binding studies with structurally diverse P2X4 receptor antagonists revealed different allosteric binding sites indicating that the new class of P2X4 receptor antagonists, to which PSB-OR-2020 belongs, interacts with an unprecedented allosteric site. [3H]PSB-OR-2020 may become a useful tool for research on P2X4 receptors and for promoting drug development.

Unraveling binding mechanism and kinetics of macrocyclic Gαq protein inhibitors.

G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gαq protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the "lipophilic anchors" of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development.

Structural response of G protein binding to the cyclodepsipeptide inhibitor FR900359 probed by NMR spectroscopy.

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of Gq proteins by blocking GDP/GTP exchange. The inhibitor binding site of Gq has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects.

Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists Binding to an Unprecedented Allosteric Site as Evidenced by Cryo-Electron Microscopy.

The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough. Aiming to develop novel, selective P2X3R antagonists, tetrazolopyrimidine-based hit compound 9 was optimized through structure-activity relationship studies by modifying the tetrazole core as well as side chain substituents. The optimized antagonist 26a, featuring a cyclopropane-substituted triazolopyrimidine core, displayed potent P2X3R-antagonistic activity (IC50 = 54.9 nM), 20-fold selectivity versus the heteromeric P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive P2X3R blockade was experimentally confirmed by calcium influx assays. Cryo-electron microscopy revealed that 26a stabilizes the P2X3R in its desensitized state, acting as a molecular barrier to prevent ions from accessing the central pore. In vivo studies in a rat neuropathic pain model (spinal nerve ligation) showed dose-dependent antiallodynic effects of 26a, thus presenting a novel, promising lead structure.

Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates.

Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry; however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered by their rapid in vivo metabolism. Although a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. This operationally simple approach provides access to pure stereoisomers of nucleoside α-thiodiphosphates and α-thiotriphosphates, as well as symmetrical or unsymmetrical dinucleoside thiodiphosphates and thiotriphosphates (including RNA cap reagents). We demonstrate that ligand-receptor interactions can be dramatically influenced by P-stereochemistry, showing that such thioisosteric replacements can have profound effects on the potency and stability of lead candidates.

L-Citrulline supplementation attenuates aortic pulse pressure and wave reflection responses to cold stress in older adults.

BACKGROUND AND AIMS: Augmented aortic systolic blood pressure (SBP) and wave reflection via sympathetic-mediated vasoconstriction elevates the risk for adverse cardiovascular events in older adults. L-citrulline (L-CIT) supplementation has shown to reduce aortic SBP and pulse pressure (PP) responses to cold pressor test (CPT) induced sympathoactivation in young men. The aim of this study was to elucidate the efficacy of L-CIT supplementation to attenuate aortic hemodynamic responses to CPT in older adults. METHODS AND RESULTS: Sixteen older adults were randomly assigned to placebo or L-CIT (6 g/day) for 14-days in a crossover, double-blind, placebo-controlled design. Brachial SBP and aortic SBP, PP, augmented pressure (AP), augmentation index standardized at 75 bpm (AIx@75), and pressure of the forward (Pf) and reflected (Pb) waves were evaluated at rest and during CPT pre- and post-intervention. Although no hemodynamic changes at rest, brachial SBP (Δ-12 ± 18 vs. Δ4 ± 14 mmHg; P = 0.008) and aortic SBP (Δ-10 ± 14 vs. Δ4 ± 12 mmHg; P = 0.005), PP (Δ-10 ± 12 vs. Δ4 ± 11 mmHg; P = 0.002), AP (Δ-4 ± 4 vs. Δ2 ± 7 mmHg; P = 0.004), AIx@75 (Δ-3.2 ± 7.2 vs. Δ2.2 ± 6.9%; P = 0.038), Pf (Δ-6 ± 10 vs. Δ3 ± 9 mmHg; P = 0.019), and Pb (Δ-4 ± 6 vs. Δ2 ± 6 mmHg; P = 0.008) responses to the CPT were significantly attenuated following L-CIT supplementation vs. placebo. CONCLUSIONS: L-CIT supplementation attenuated aortic pulsatile pressure and pressure wave reflection responses to CPT in older adults, providing possible cardioprotection during cold-induced sympathoactivation in older adults.

Extracellular binding sites of positive and negative allosteric P2X4 receptor modulators.

AIMS: P2X receptors are ATP-gated ion channels which play a role in many pathophysiological conditions. They are considered as novel drug targets, particularly in the fields of pain, (neuro) inflammation, and cancer. Due to difficulties in developing drug-like orthosteric ligands that bind to the highly polar ATP binding site, the design of positive and negative allosteric modulators (PAMs and NAMs) is a promising strategy. The P2X4 receptor was proposed as a novel target for neuropathic and inflammatory pain (antagonists), and for the treatment of alcoholism (PAMs). So far, little is known about the allosteric binding site(s) of P2X4 receptors. The aim of this study was to identify the binding site(s) of the macrocyclic natural product ivermectin, the urea derivative BX430, and the antidepressant drug paroxetine that act as allosteric modulators of P2X4 receptors. MATERIAL AND METHODS: We generated chimeric receptors in which extracellular sequences of the human P2X4 receptor were exchanged for corresponding residues of the human P2X2 receptor, complemented by specific single amino acid residue mutants. Chimeric and mutated receptors were stably expressed in 1321N1 astrocytoma cells, and characterized by fluorimetric measurement of ATP-induced Ca2+-influx. In addition, docking studies utilizing a homology model of the human P2X4 receptor were performed. KEY FINDINGS: Our results suggest a common binding site for ivermectin and BX430 in an extracellular receptor domain, while paroxetine might bind to the cation pore. SIGNIFICANCE: The obtained results provide a basis for the development of positive and negative allosteric P2X4 modulators with improved properties and will support future drug development efforts.

Crossroads of highly pathogenic H5N1: overlap between wild and domestic birds in the Black Sea-Mediterranean impacts global transmission.

Understanding transmission dynamics that link wild and domestic animals is a key element of predicting the emergence of infectious disease, an event that has highest likelihood of occurring wherever human livelihoods depend on agriculture and animal trade. Contact between poultry and wild birds is a key driver of the emergence of highly pathogenic avian influenza (HPAI), a process that allows for host switching and accelerated reassortment, diversification, and spread of virus between otherwise unconnected regions. This study addresses questions relevant to the spillover of HPAI at a transmission hotspot: what is the nature of the wild bird-poultry interface in Egypt and adjacent Black Sea-Mediterranean countries and how has this contributed to outbreaks occurring worldwide? Using a spatiotemporal model of infection risk informed by satellite tracking of waterfowl and viral phylogenetics, this study identified ecological conditions that contribute to spillover in this understudied region. Results indicated that multiple ducks (Northern Shoveler and Northern Pintail) hosted segments that shared ancestry with HPAI H5 from both clade 2.2.1 and clade 2.3.4 supporting the role of Anseriformes in linking viral populations in East Asia and Africa over large distances. Quantifying the overlap between wild ducks and H5N1-infected poultry revealed an increasing interface in late winter peaking in early spring when ducks expanded their range before migration, with key differences in the timing of poultry contact risk between local and long-distance migrants.

Low Pathogenic Avian Influenza Viruses in Wild Migratory Waterfowl in a Region of High Poultry Production, Delmarva, Maryland.

Migratory waterfowl are natural reservoirs for low pathogenic avian influenza viruses (AIVs) and may contribute to the long-distance dispersal of these pathogens as well as spillover into domestic bird populations. Surveillance for AIVs is critical to assessing risks for potential spread of these viruses among wild and domestic bird populations. The Delmarva Peninsula on the east coast of the United States is both a key convergence point for migratory Atlantic waterfowl populations and a region with high poultry production (>4,700 poultry meat facilities). Sampling of key migratory waterfowl species occurred at 20 locations throughout the Delmarva Peninsula in fall and winter of 2013-14. Samples were collected from 400 hunter-harvested or live-caught birds via cloacal and oropharyngeal swabs. Fourteen of the 400 (3.5%) birds sampled tested positive for the AIV matrix gene using real-time reverse transcriptase PCR, all from five dabbling duck species. Further characterization of the 14 viral isolates identified two hemagglutinin (H3 and H4) and four neuraminidase (N2, N6, N8, and N9) subtypes, which were consistent with isolates reported in the Influenza Research Database for this region. Three of 14 isolates contained multiple HA or NA subtypes. This study adds to the limited baseline information available for AIVs in migratory waterfowl populations on the Delmarva Peninsula, particularly prior to the highly pathogenic AIV A(H5N8) and A(H5N2) introductions to the United States in late 2014.

Decreasing body dissatisfaction using a brief conditioning intervention.

OBJECTIVE: Body dissatisfaction in females is common and a risk factor for the development of an eating disorder. This study tested whether body dissatisfaction could be improved using a brief conditioning intervention in which photographs of participants' bodies were selectively paired with positive social stimuli (smiling faces) and photographs of other bodies were paired with neutral or negative social stimuli (neutral and frowning faces). METHOD: 39 women (mean age = 22.46; 64.1% Caucasian) with high body dissatisfaction were randomized to either the evaluative conditioning intervention (n = 22) or to a delayed waitlist control condition (n = 17). Body dissatisfaction (specifically, shape and weight concern), restraint, eating concern, and self-esteem were assessed at baseline, post treatment and again after four and 12 weeks. RESULTS: Compared to women in the delayed waitlist control condition, women in the treatment condition demonstrated a significant decrease in shape and weight concern, and a significant increase in self-esteem. Similar trends were found for the control condition after they completed the intervention. Changes at post treatment related to body dissatisfaction were maintained at 12-week follow-up. CONCLUSIONS: Repeatedly pairing photographs of an individual's body with positive social feedback may lead to improved body image and self-esteem.