Divergent Effects of the Purinoceptor Antagonists Suramin and Pyridoxal-5 -phosphate-6-(2 -naphthylazo-6 -nitro-4 ,8 - disulfonate) (PPNDS) on -Amino-3-hydroxy-5-methyl-4- isoxazolepropionic Acid (AMPA) Receptors

Suramin is a large naphthyl-polysulfonate compound that inhibits an array of receptors and enzymes, and it has also been reported to block currents mediated by glutamate receptors. This study shows that suramin and several structurally related compounds [8,8 -[carbonylbis(imino-3,1-phenylenecarbonylamino)]bis-(1,3,5naphthalenetrisulfonic acid), 6Na (NF023), 8,8 -(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis-1,3,5-naphthalenetrisulfonic acid, Na (NF279), and 4,4 ,4 ,4 -[carbonyl-bis[imino-5,1,3-benzenetriyl-bis-(carbonylimino)]]tetrakis-benzene-1,3-disulfonic acid, 8Na (NF449)] reduce binding of [H] -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and [H]fluorowillardiine to rat brain membranes and homomeric GluR1–4 receptors, with IC50 values in the range of 5 to 180 M. Inhibition often was less than complete at saturating drug concentrations and thus seems to be noncompetitive in nature. Pyridoxal-5 -phosphate-6-(2 -naphthylazo-6 -nitro-4 ,8 disulfonate) (PPNDS) is a potent antagonist of purinoceptors that shares some structural elements with suramin yet is smaller than the latter. PPNDS also had potent effects on AMPA receptors (EC50 value of 4 M) but of a kind not seen with the other compounds in that it increased binding affinity for radioagonists severalfold. In addition, PPNDS slowed association and dissociation rates more than 10 times. In physiological experiments with GluR2 receptors, PPNDS at 50 M reduced the peak current by 30 to 50% but had only small effects on other waveform aspects such desensitization and steady-state currents. This pattern of effects differentiates PPNDS from other compounds such as thiocyanate and up-modulators, which increase agonist binding by enhancing desensitization or slowing deactivation, respectively. Receptor model simulations indicate that most effects can be accounted for by assuming that PPNDS slows agonist binding/ unbinding and stabilizes the bound-closed state of the receptor. By extension, suramin is proposed to stabilize the unbound state and thereby to reduce affinity for agonists. These drugs thus act through a novel type of noncompetitive antagonism. Suramin is an extended molecule with a symmetrical backbone of amide-linked aromatic rings and with three negatively charged sulfonate substituents on each of the terminal naphthyl elements (Fig. 1). As such, it shares many structural similarities with a broader class of polysulfonated compounds that include the azo-dyes trypan blue, Evans blue, Chicago sky blue, and basilen blue (also called reactive blue 2). Like many of the latter, suramin has a remarkably diverse range of pharmacological actions, yet the mechanisms underlying these effects are in most instances still obscure. Suramin was developed 80 years ago and is still occasionally used for the treatment of trypanosomiasis and onchocerciasis (Wang 1995). In vitro, suramin affects a bewildering array of molecular and cellular processes. It inhibits numerous signaling proteins, including growth factors and interleukins (Voogd et al., 1993); among its many other targets are G proteins (Freissmuth et al., 1996) and enzymes such as reverse transcriptase (De Clercq, 1987) and ectonucleotidases This work was supported by National Institutes of Health grant NS41020. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.003038. ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate; PPADS, pyridoxal phosphate-6azophenyl-2 ,4 -disulfonic acid; CNQX, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline; PPNDS, pyridoxal-5 -phosphate-6-(2 -naphthylazo-6 -nitro-4 ,8 disulfonate); HEK, human embryonic kidney; HBS, HEPES-buffered saline; SCN, thiocyanate anion; GluR, glutamate receptor; FW, fluorowillardiine; NF023, 8,8 -[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-(1,3,5-naphthalenetrisulfonic acid), 6Na; NF279, 8,8 -(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis-1,3,5-naphthalenetrisulfonic acid, Na; NF449, 4,4 ,4 ,4 -[carbonyl-bis[imino-5,1,3-benzenetriyl-bis(carbonylimino)]]tetrakis-benzene-1,3-disulfonic acid, 8Na; GYKI52466, 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzeneamine dihydrochloride. 0026-895X/04/6606-1738–1747$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 6 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3038/1186560 Mol Pharmacol 66:1738–1747, 2004 Printed in U.S.A. 1738 at A PE T Jornals on M ay 3, 2016 m oharm .aspeurnals.org D ow nladed from (Lambrecht, 2000). However, suramin seems particularly potent in blocking purinergic receptors with low micromolar inhibition constants (Charlton et al., 1996; Lambrecht, 2000). There are also reports that suramin inhibits glutamatergic synaptic transmission (Motin and Bennett, 1995; Gu et al., 1998) and whole-cell currents mediated by AMPA receptors (Nakazawa et al., 1995; Zona et al., 2000) and NMDA receptors (Ong et al., 1997; Peoples and Li, 1998). Suramin was observed to inhibit binding of radioligands to NMDA receptors (Balcar et al., 1995) but not kainate receptors (Ong et al., 1997), which suggested some selectivity among glutamate receptors. Apart from these studies, there have been few efforts to characterize the molecular actions of suramin and the nature of the impact on glutamate receptor function. Even less is known about possible interactions with more recent P2X receptor antagonists, many of which possess structural elements similar to those in suramin. Figure 1 shows examples of three such compounds with terminal naphthyl-polysulfonate groups (Freissmuth et al., 1996; Lambrecht, 2000; Rettinger et al., 2000). A somewhat distinct structural family of purinoceptor antagonists was established some time ago with the development of PPADS (Lambrecht et al., 1992), which has since been widely used as a highly selective P2X receptor blocker. This compound does not have mirror symmetry, is much smaller than suramin, and was indeed found to have no or weak effects on glutamate receptor responses (Motin and Bennett, 1995; Gu et al., 1998; Zona et al., 2000). PPNDS, another member of this drug family, has recently been identified as having even greater affinity for P2X receptors and a marked selectivity toward the P2X1 receptor subtype with a nanomolar inhibition constant (Lambrecht et al., 2000). Yet the two sulfonate groups and the nitro substituent in this compound occupy the same positions as the sulfonate groups in suramin (Fig. 1) and thus the possibility of an interaction with glutamate receptors cannot be dismissed. The present study was intended to examine whether suramin inhibits binding to AMPA receptors, to clarify the nature of such inhibition, and to test whether the structurally related compounds have similar actions. Binding tests with radiolabeled agonists were used initially because they can readily distinguish between different subclasses of antagonists and their mode of inhibition. For instance, if suramin were to act as a competitive antagonist then it should completely displace radiolabeled agonists such as [H]AMPA from their binding site, as it is the case for quinoxaline compounds such as CNQX (Honore and Drejer, 1988). In contrast, 2,3-benzodiazepines such as GYKI52466 block AMPA receptor currents through a distinct site because they do not inhibit the binding of agonists (Kessler et al., 1996), except under specific circumstances (Arai et al., 2002b). Guanine nucleotides constitute a third group of AMPA receptor blockers that also reduce agonist binding but through an as yet unidentified mechanism (Monahan et al., 1988; Baron et al., 1989; Dev et al., 1996). Last, if suramin were to act as a channel blocker then binding of agonists would presumably remain unaffected. During examination of the structural analogs described above, it was also found that PPNDS affected AMPA receptors in ways that differed substantially from those of suramin, both in physiological and binding experiments. Thus, the second goal of this project was to identify which aspects of receptor kinetics are most likely influenced by PPNDS. The findings with both suramin and PPNDS have been incorporated into a working model that suggests that both drugs bind to a domain near the agonist-binding site yet with opposite consequences for the stability of the agonist-bound state. Materials and Methods Binding Assays. Binding tests were carried out with membranes from rat brain and from HEK293 cells expressing one of the AMPA receptor subunits. Animals were anesthetized with halothane before decapitation according to an institutionally approved protocol and in observation of the guidelines of the National Institutes of Health. Fig. 1. Drug structures. For drugs that possess mirror symmetry, only one-half the molecule is shown; the symmetry axis is indicated with a dashed line. PPADS and PPNDS are derivatives of pyridoxal-5-phosphate and do not have mirror-symmetry. Evans blue has been reported to inhibit AMPA receptor currents (Keller et al., 1993). All compounds are normally supplied as sodium salts. The formulas show the anionic forms prevailing in neutral aqueous solutions. PPNDS Effects on AMPA Receptors 1739 at A PE T Jornals on M ay 3, 2016 m oharm .aspeurnals.org D ow nladed from Membranes from rat brain were prepared according to conventional procedures (Kessler et al., 1996) involving homogenization in isotonic sucrose and differential centrifugation to obtain a P2 pellet fraction, followed by an osmotic lysis and repeated washing by centrifugation. For binding tests, membranes were suspended in HEPES-buffered saline (HBS; 150 mM NaCl, 20 mM HEPES, and 0.1 mM EGTA, pH 7.4). Binding studies with recombinant receptors used a preparation of permeabilized and washed cells. Most receptor subunits examined here are stably expressed in HEK293 cells (Hennegriff et al., 1997; Arai et al., 2000) and periodically confirmed by Western blots. HEK293 cells were collected into HBS containing in addition 2 mM EGTA and a protease inhibitor cocktail (SigmaAldrich, St. Louis, MO). The cells were centrifuged at least four times (2000g; 10 min) and resuspended in HBS without additions; after the first centrifugation, 0.1% saponin was included in the HBS to permeabilize the membranes, and the cell suspension was left at 25°C for 3 min. All other steps were carried out at 0 to 4°C. The cells were stored on ice for up to 1 month without evident change in receptor properties and washed on each test day before use. For some tests, the permeabilized cells were extensively sonicated with an ice-cooled tip, and the resulting membrane fragments were collected by highspeed centrifugation (15 min; 60,000g). Unless mentioned otherwise, binding tests were conducted using the following protocols. Aliquots of rat brain membranes or permeabilized cells (10–20 g of protein) were incubated at 0°C with radioligand and appropriate additions in 50l volume. Where indicated ( SCN), incubation media contained 50 mM KSCN (potassium thiocyanate), which increases affinity for [H]AMPA about 10-fold. Incubations were terminated after 30 to 60 min by filtration through GF/A or GF/C filters after diluting the sample in 5 ml of ice-cold buffered saline containing 50 mM KSCN (wash buffer); the filters were rapidly rinsed with 15 ml of additional wash buffer. In some assays with brain membranes, incubations were terminated by 15min centrifugation in a Beckman JA 18.1 rotor with rotor temperature calibrated to match incubation temperature. The pellet was quickly rinsed with ice-cold wash buffer. Stock solutions of test drugs were prepared in HBS. Nonspecific binding was determined with 5 mM glutamate and subtracted from total binding. The drugs in general had no significant effect on nonspecific binding and did not cause quenching of scintillation counting. Protein content was determined according to Bradford (1976) with serum albumin as standard. Binding data were usually normalized and averaged across experiments and plotted as mean and S.E.M. Binding curves were fitted through nonlinear regression using the Prism program from GraphPad Software Inc. (San Diego, CA). Excised Patch Currents Mediated by GluR2 Receptors. Currents mediated by homomeric GluR2(R607Q) flop or flip receptors were measured in HEK293 cells transiently transfected with the respective cDNAs in a pRK5 vector, using LipofectAMINE (Invitrogen, Carlsbad, CA). Cells were submitted to experimentation 72 to 96 h after transfection. Patch pipettes had a resistance of 2 to 5 M and were filled with a solution of 130 mM CsF, 10 mM EGTA, 2 mM ATP Mg salt, and 10 mM HEPES, pH 7.3. The extracellular solution contained 140 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.3. Patches were excised from the cells and positioned in front of a double pipet delivering a constant flow of background medium (without or with test drug) in one flow line and medium containing 1 mM L-glutamate in the second flow line. The patch was initially placed in the background flow line. A piezo device then moved the double pipet in a fraction of a millisecond into the glutamate flow line (Arai et al., 1996). In general, 10 responses were collected at 5-s intervals and averaged to give one trial. When testing PPNDS, several control trials were collected and then the background flow line was changed to medium containing 50 M PPNDS. After three to four drug trials, the background medium was switched back to control medium. In tests with suramin, trials were repeatedly alternated between control condition and the various drug concentrations. The holding potential was 70 mV. Data were acquired with a patch amplifier (AxoPatch-1D) at a filter frequency of 5 kHz and digitized at 10 kHz with PClamp/Digidata 1322A (Axon Instruments Inc., Union City,