Sean G. Brown

Selectivity of diadenosine polyphosphates for rat P2X receptor subunits.

The pharmacological activity of diadenosine polyphosphates was investigated at three recombinant P2X receptors (rat P2X1, rat P2X3, rat P2X4) expressed in Xenopus oocytes and studied under voltage-clamp conditions. For the rat P2X1 receptor, only P1,P6-diadenosine hexaphosphate (Ap6A) was a full agonist yet 2-3 folds less potent than ATP. At rat P2X3, P1,p4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A) and Ap6A were full agonists and more potent than ATP. Ap4A alone was equipotent with ATP at rat P2X4, but only as a partial agonist. Compared to known data for rat P2X2 and human P2X1 receptors, our findings contrast with rat P2X2 where only Ap4A is a full agonist although four folds less potent than ATP. At rat and human orthologues of P2X1, Ap5A was a partial agonist with similar potency. These data provide a useful basis for selective agonists of P2X receptor subunits.

Activity of novel adenine nucleotide derivatives as agonists and antagonists at recombinant rat P2X receptors

The effects of structural modifications of adenine nucleotides previously shown to enhance either agonist (2‐thioether groups) or antagonist (additional phosphate moieties at the 3′‐ or 2′‐position) properties at P2Y1 receptors were examined at recombinant rat P2X1, P2X2, P2X3, and P2X4 receptors expressed in Xenopus oocytes. The potency of P2Y1 agonists HT‐AMP (2‐(hexylthio)adenosine‐5′‐monophosphate) and PAPET (2‐[2‐(4‐aminophenyl)ethylthio]adenosine‐5′‐triphosphate) was examined at P2X receptors. Both nucleotides showed a preference for the Group I (α,β‐meATP‐sensitive, fast‐inactivating) P2X subunits. HT‐AMP was 5‐fold more potent than ATP at P2X3 receptors and a partial agonist at all except P2X2 receptors, at which it was a full agonist. The efficacy of HT‐AMP was as low as 23% at P2X4 receptors. PAPET was a weak partial agonist at rat P2X4 receptors and a nearly full agonist at the other subtypes. At rat P2X3 receptors, PAPET was more potent than any other known agonist (EC50 = 17 ± 3 nM). MRS 2179 (N6‐methyl‐2′‐deoxyadenosine 3′, 5‐bisphosphate, a potent P2Y1 receptor antagonist) inhibited ATP‐evoked responses at rat P2X1 receptors with an IC50 value of 1.15 ± 0.21 μM. MRS 2179 was a weak antagonist at rat P2X3 receptors, with an IC50 value of 12.9 ± 0.1 μM, and was inactive at rat P2X2 and P2X4 receptors. Thus, MRS 2179 was 11‐fold and 130‐fold selective for P2Y1 receptors vs. P2X1 and P2X3 receptors, respectively. MRS 2209, the corresponding 3′‐deoxy‐2′‐phosphate isomer, was inactive at rat P2X1 receptors, thus demonstrating its greater selectivity as a P2Y1 receptor antagonist. Various adenine bisphosphates in the family of MRS 2179 containing modifications of either the adenine (P2Y1 antagonists with 2‐ and 6‐substitutions), the phosphate (a 3′,5′‐cyclic diphosphate, inactive at P2Y1 receptors), or the ribose moieties (antagonist carbocyclic analogue), were inactive at both rat P2X1 and P2X3 receptors. An anhydrohexitol derivative (MRS 2269) and an acyclic derivative (MRS 2286), proved to be selective antagonists at P2Y1 receptors, since they were inactive as agonist or antagonist at P2X1 and P2X3 receptors. Drug Dev. Res. 49:253–259, 2000. Published 2000 Wiley‐Liss, Inc.

Molecular recognition in P2 receptors: ligand development aided by molecular modeling and mutagenesis.

Publisher Summary As molecular modeling of cloned G protein-coupled receptor (GPCR) sequences using a rhodopsin template has been refined, it has become possible to generate hypotheses for location of the binding sites that are consistent with mutagenesis results and ligand specificities. To obtain an energetically refined 3-D structure of the ligand–receptor complex, the chapter introduces a new computational approach, a “cross docking” procedure, which simulates the reorganization of the native receptor induced by the ligand. The molecular basis for recognition by human P2Y 1 receptors of the selective, competitive antagonist MRS 2179 is probed using site-directed mutagenesis and molecular modeling. The model was derived from primary sequence comparison, secondary structure predictions, and 3-D homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. A putative nucleotide binding site was localized, following a cross docking procedure to obtain energetically refined 3-D structures of the ligand–receptor complexes, and used to predict which residues are likely to be in proximity to agonists and antagonists. Molecular modeling using PowerFit has suggested a possible model of superimposition of two classes of antagonists, nucleotides related to MRS 2179, and non-nucleotides related to pyridoxal phosphate.

Differential Phosphoinositide Binding to Components of the G Protein-Gated K+ Channel

The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K+ channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.