Ionotropic purinergic receptor 7 (P2X7) channel structure and pharmacology provides insight regarding non-nucleotide agonism.

P2X7 is a member of the Ionotropic Purinergic Receptor (P2X) family. The P2X family of receptors is composed of seven (P2X1-7), ligand-gated, nonselective cation channels. Changes in P2X expression have been reported in multiple disease models. P2Xs have large complex extracellular domains that function as receptors for a variety of ligands, including endogenous and synthetic agonists and antagonists. ATP is the canonical agonist. ATP affinity ranges from nanomolar to micromolar for most P2XRs, but P2X7 has uniquely poor ATP affinity. In many physiological settings, it may be difficult to achieve the millimolar extracellular ATP concentrations needed for P2X7 channel activation; however, channel function is implicated in pain sensation, immune cell function, cardiovascular disease, cancer, and osteoporosis. Multiple high-resolution P2X7 structures have been solved in apo-, ATP-, and antagonist-bound states. P2X7 structural data reveal distinct allosteric and orthosteric antagonist-binding sites. Both allosteric and orthosteric P2X7 antagonists are well documented to inhibit ATP-evoked channel current. However, a growing body of evidence supports P2X7 activation by non-nucleotide agonists, including extracellular histone proteins and human cathelicidin-derived peptides (LL-37). Interestingly, P2X7 non-nucleotide agonism is not inhibited by allosteric antagonists, but is inhibited by orthosteric antagonists. Herein, we review P2X7 function with a focus on the efficacy of available pharmacology on P2X7 channel current activation by non-nucleotide agonists in effort to understand agonist/antagonist efficacy, and consider the impact of these data on the current understanding of P2X7 in physiology and disease given these limitations of P2X7-selective antagonists and incomplete knockout mouse models.

Extracellular histone proteins activate P2XR7 channel current.

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

L-Citrulline Supplementation Increases Plasma Nitric Oxide Levels and Reduces Arginase Activity in Patients With Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is becoming a major contributor to cardiovascular disease. One of the early signs of T2DM associated cardiovascular events is the development of vascular dysfunction. This dysfunction has been implicated in increasing the morbidity and mortality of T2DM patients. One of the important characteristics of vascular dysfunction is the impaired ability of endothelial cells to produce nitric oxide (NO). Additionally, decreases in the availability of NO is also a major contributor of this pathology. NO is produced by the activity of endothelial NO synthase (eNOS) on its substrate, L-arginine. Reduced availability of L-arginine to eNOS has been implicated in vascular dysfunction in diabetes. Arginase, which metabolizes L-arginine to urea and ornithine, competes directly with NOS for L-arginine. Hence, increases in arginase activity can decrease arginine levels, reducing its availability to eNOS and decreasing NO production. Diabetes has been linked to elevated arginase and associated vascular endothelial dysfunction. We aimed to determine levels of plasma NO and arginase activity in (T2DM) patients and the effects of L-citrulline supplementation, a natural arginase inhibitor, on inhibiting arginase activity in these patients. Levels of arginase correlated with HbA1c levels in diabetic patients. Twenty-five patients received L-citrulline supplements (2000 mg/day) for 1 month. Arginase activity decreased by 21% in T2DM patients after taking L-citrulline supplements. Additionally, plasma NO levels increased by 38%. There was a modest improvement on H1Ac levels in these patients, though not statistically significant. The effect of L-citrulline on arginase activity was also studied in bovine aortic endothelial cells (BAECs) grown in high glucose (HG) conditions. HG (25 mM, 72 h) caused a 2-fold increase in arginase activity in BAECs and decreased NO production by 30%. L-citrulline (2.5 mM) completely prevented the increase in arginase activity and restored NO production levels. These data indicate that L-citrulline can have therapeutic benefits in diabetic patients through increasing NO levels and thus maintaining vascular function possibly through an arginase inhibition related pathway.

Discovery of New Phosphoinositide 3-kinase Delta (PI3Kδ) Inhibitors via Virtual Screening using Crystallography-derived Pharmacophore Modelling and QSAR Analysis.

BACKGROUND: PI3Kδ is predominantly expressed in hematopoietic cells and participates in the activation of leukocytes. PI3Kδ inhibition is a promising approach for treating inflammatory diseases and leukocyte malignancies. Accordingly, we decided to model PI3Kδ binding. METHODS: Seventeen PI3Kδ crystallographic complexes were used to extract 94 pharmacophore models. QSAR modelling was subsequently used to select the superior pharmacophore(s) that best explain bioactivity variation within a list of 79 diverse inhibitors (i.e., upon combination with other physicochemical descriptors). RESULTS: The best QSAR model (r2 = 0.71, r2 LOO = 0.70, r2 press against external testing list of 15 compounds = 0.80) included a single crystallographic pharmacophore of optimal explanatory qualities. The resulting pharmacophore and QSAR model were used to screen the National Cancer Institute (NCI) database for new PI3Kδ inhibitors. Two hits showed low micromolar IC50 values. CONCLUSION: Crystallography-based pharmacophores were successfully combined with QSAR analysis for the identification of novel PI3Kδ inhibitors.

Ligand-based computational modelling of platelet-derived growth factor beta receptor leading to new angiogenesis inhibitory leads.

Platelet derived growth factor beta receptor (PDGFR- ß) plays an important role in angiogenesis. PDGFR-ß expression is correlated with increased vascularity and maturation of blood vessels in cancer. Pharmacophore modeling and quantitative structure-activity relationship (QSAR) analysis were combined to explore the structural requirements for ligand-PDGFR-ß recognition using 107 known PDGFR-ß inhibitors. Genetic function algorithm (GFA) coupled to k nearest neighbor (kNN) and multiple linear regression (MLR) analysis were employed to generate predictive QSAR models based on optimal combinations of pharmacophores and physicochemical descriptors. The successful pharmacophores were complemented with exclusion spheres to optimize their receiver operating characteristic curve (ROC) profiles. The QSAR models and their associated pharmacophore hypotheses were validated by identification and experimental evaluation of new angiogenesis inhibitory leads retrieved from the National Cancer Institute (NCI) structural database. Two hits illustrated low micromolar IC50 values in two distinct anti-angiogenesis bioassays.

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CLpro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CLpro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CLpro . HKU4-CoV 3CLpro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CLpro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CLpro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CLpro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CLpro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CLpro . HIGHLIGHTS: MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CLpro is a potential drug target for coronavirus infection. HKU4-CoV 3CLpro is a useful surrogate model for the identification of MERS-CoV 3CLpro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CLpro inhibitors development.