PET imaging of P2X7R in the experimental autoimmune encephalomyelitis model of multiple sclerosis using [11C]SMW139.

BACKGROUND: Non-invasive imaging of the activation status of microglia and the ability to identify a pro- or anti-inflammatory environment can provide valuable insights not only into pathogenesis of neuro-inflammatory and neurodegenerative diseases but also the monitoring of the efficacy of immunomodulatory therapies. P2X7R is highly expressed on pro-inflammatory microglia and [11C]SMW139, a specific P2X7R tracer for positron emission tomography imaging, showed good pharmacokinetics, stability, and brain permeability in vivo. Our objective was to evaluate the potential of [11C]SMW139 for PET imaging of neuroinflammation in vivo in the experimental autoimmune encephalomyelitis (EAE) model. METHODS: We induced EAE in Lewis rats by immunization with MBP 69-88 in complete Freund's adjuvant (CFA). We determined the affinity of [11C]SMW139 to human and rat P2X7R using saturation binding assay. Using this tracer, PET imaging was performed at the peak of disease and in the recovery phase. In vivo blocking experiments were conducted to validate the specific brain uptake of the tracer. Immunohistochemistry staining and autoradiography were performed to evaluate the level of neuroinflammation and validate the specific binding of [11C]SMW139. RESULTS: [11C]SMW139 showed good affinity for the rat P2X7R with a Kd of 20.6 ± 1.7 nM. The uptake of [11C]SMW139 was significantly higher in EAE animals at the peak of disease compared to the recovery phase but not in CFA control animals. The amplitude of increase of [11C]SMW139 uptake showed significant positive correlation with clinical scores mainly in the spinal cord (Pearson = 0.75, Spearman = 0.76; p < 0.0001). Treating EAE animals with P2X7R antagonist JNJ-47965567 blocked the uptake of [11C]SMW139 in the spinal cord, cerebellum, and brain stem, demonstrating specific accumulation of the tracer. P-glycoprotein blocking with tariquidar (30 mg/kg) did not affect tracer penetration in the brain showing that [11C]SMW139 is not a Pgp substrate. CONCLUSION: Our data shows that [11C]SMW139 is a promising PET tracer for imaging neuroinflammation and evaluating the dynamics of pro-inflammatory microglia in the brain. This can provide crucial insights into the role of microglia in disease progression and enables the development of novel treatment strategies aimed at modulating the immune response in order to promote neuroprotection.

Prevention and rescue of cardiac dysfunction by methanocarba adenosine monophosphonate derivatives.

Accumulating evidence supports a therapeutic role of purinergic signaling in cardiac diseases. Previously, efficacy of systemically infused MRS2339, a charged methanocarba derivative of 2-Cl-adenosine monophosphate, was demonstrated in animal models of heart failure. We now test the hypothesis that an uncharged adenine nucleoside phosphonate, suitable as an oral agent with a hydrolysis-resistant phospho moiety, can prevent the development of cardiac dysfunction in a post-infarction ischemic or pressure overload-induced heart failure model in mice. The diester-masked uncharged phosphonate MRS2978 was efficacious in preventing cardiac dysfunction with improved left ventricular (LV) fractional shortening when administered orally at the onset of ischemic or pressure overload-induced heart failure. MRS2925, the charged, unmasked MRS2978 analog, prevented heart dysfunction when infused subcutaneously but not by oral gavage. When administered orally or systemically, MRS2978 but not MRS2925 could also rescue established cardiac dysfunction in both ischemic and pressure overload heart failure models. The diester-masked phosphate MRS4074 was highly efficacious at preventing the development of dysfunction as well as in rescuing pressure overload-induced and ischemic heart failure. MRS2978 was orally bioavailable (57-75%) giving rise to MRS2925 as a minor metabolite in vivo, tested in rats. The data are consistent with a novel therapeutic role of adenine nucleoside phosphonates in systolic heart failure.

Molecular determinants for agonist recognition and discrimination in P2X2 receptors.

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.

Purinergic P2X receptors: structural models and analysis of ligand-target interaction.

The purinergic P2X receptors are ligand-gated cation channels activated by the endogenous ligand ATP. They assemble as homo- or heterotrimers from seven cloned subtypes (P2X1-7) and all trimer subunits present a common topology consisting in intracellular N- and C- termini, two transmembrane domains and a large extracellular domain. These membrane proteins are present in virtually all mammalian tissues and regulate a large variety of responses in physio- and pathological conditions. The development of ligands that selectively activate or block specific P2X receptor subtypes hence represents a promising strategy to obtain novel pharmacological tools for the treatment of pain, cancer, inflammation, and neurological, cardiovascular, and endocrine diseases. The publication of the crystal structures of zebrafish P2X4 receptor in inactive and ATP-bound active forms provided structural data for the analysis of the receptor structure, the interpretation of mutagenesis data, and the depiction of ligand binding and receptor activation mechanism. In addition, the availability of ATP-competitive ligands presenting selectivity for P2X receptor subtypes supports the design of new potent and selective ligands with possibly improved pharmacokinetic profiles, with the final aim to obtain new drugs. This study describes molecular modelling studies performed to develop structural models of the human and rat P2X receptors in inactive and active states. These models allowed to analyse the role of some non-conserved residues at ATP binding site and to study the receptor interaction with some non-specific or subtype selective agonists and antagonists.

Medicinal chemistry of P2X receptors: agonists and orthosteric antagonists.

In this work, we have highlighted data reported in the literature trying to draw a complete picture of the structures and biological activity of agonists and orthosteric antagonists of P2X receptors. Actually, only few P2X receptor agonists have been found and most of them are derived from modification of the natural ligand ATP and they are P2X receptor subtype unselective. In particular, BzATP (9) is one of the most potent P2X receptor agonists with EC50 value in the nanomolar range at some subtypes. Differently from agonists, P2X receptor antagonists belong to different chemical classes such as high molecular weight aryl polysulfonate molecules like suramin and its simplified derivatives and anthraquinone compounds. All these molecules proved to be non selective at P2X receptors, and they are endowed with micromolar activity and not favourable pharmacokinetic properties due to the presence of several charged groups. Also modification of the natural ligand ATP led to the discovery of P2X receptor antagonists like TNP-ATP (29), which, although not selective, showed high potency at P2X1, P2X3 (IC50 of 0.006 µM and 0.001 µM, respectively), and heteromeric P2X2/3 receptors. Also the dinucleotide inosine polyphosphate Ip5I (33) was found to be a potent and selective antagonist at P2X1 vs P2X3 receptors with IC50 = 0.003 µM. A significant improvement has been gained from the interest of pharmaceutical companies that in the last years discovered, through the use of high-throughput screening, potent and selective antagonists endowed with novel structures, some of which are currently in clinical trials for several therapeutic applications.

Unanticipated parallels in architecture and mechanism between ATP-gated P2X receptors and acid sensing ion channels.

ATP-gated P2X receptors and acid-sensing ion channels are cation-selective, trimeric ligand-gated ion channels unrelated in amino acid sequence. Nevertheless, initial crystal structures of the P2X4 receptor and acid-sensing ion channel 1a in resting/closed and in non conductive/desensitized conformations, respectively, revealed common elements of architecture. Recent structures of both channels have revealed the ion channels in open conformations. Here we focus on common elements of architecture, conformational change and ion permeation, emphasizing general principles of structure and mechanism in P2X receptors and in acid-sensing ion channels and showing how these two sequence-disparate families of ligand-gated ion channel harbor unexpected similarities when viewed through a structural lens.

P2X7-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells.

The P2X7 receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X7 function exhibit altered bone formation. Moreover, activation of P2X7 in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X7 agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP; 300 µM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X7 receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 µM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (∼2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 µM), which activate P2 receptors other than P2X7, failed to elicit a sustained increase in proton efflux. Specific P2X7 receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca²âº was required during P2X7 receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X7 receptors on osteoblast-like cells triggers a dramatic, Ca²âº-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.

Amino acid residues constituting the agonist binding site of the human P2X3 receptor.

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,ß-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.