Epidemiological evidence suggests a lower incidence of Parkinson's disease in smokers than in nonsmokers. This evidence, together with the lower levels of brain monoamine oxidase (MAO) activity in smokers and the potential neuroprotective properties of MAO inhibitors, prompted studies which led to the isolation and characterization of 2,3,6-trimethyl-1,4-naphthoquinone (TMN), an MAO-A and MAO-B inhibitor which is present in tobacco and tobacco smoke. Results of experiments reported here provide evidence that this compound protects against the MPTP-mediated depletion of neostriatal dopamine levels in the C57BL/6 mouse. These results support the hypothesis that the inhibition of MAO by constituents of tobacco smoke may be related to the decreased incidence of Parkinson's disease in smokers.
Caffeine and more specific antagonists of the adenosine A2A receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A2A antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP+, we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP+ and of MPP+, suggesting that CSC blocks the conversion of MPTP to MPDP+ in vivo. In assessing the direct effects of CSC and A2A receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with aK i value of 100 nm, whereas caffeine and another relatively specific A2A antagonist produced little or no inhibition. The A2A receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A2A receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A2A receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A2A receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties. Caffeine and more specific antagonists of the adenosine A2A receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A2A antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP+, we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP+ and of MPP+, suggesting that CSC blocks the conversion of MPTP to MPDP+ in vivo. In assessing the direct effects of CSC and A2A receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with aK i value of 100 nm, whereas caffeine and another relatively specific A2A antagonist produced little or no inhibition. The A2A receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A2A receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A2A receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A2A receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties. Parkinson's disease 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine 8-(3-chlorostyryl)caffeine 1-methyl-4-phenylpyridinium monoamine oxidase adenosine A2A receptor knockout wild-type 1,3-dipropyl-8-cylcopentylxanthine 3,7-dimethyl-1-propargylxanthine 1-methyl-4-phenyl-2,3-dihydropyridinium 1-methyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydroyridine dopamine transporter The neurodegeneration of Parkinson's disease (PD)1 targets dopaminergic neurons that project to the striatum (1Lang A.E. Lozano A.M. N. Engl. J. Med. 1998; 339: 1044-1053Crossref PubMed Scopus (1773) Google Scholar). In PD the progressive loss of striatal dopamine leads to a progressive deterioration in motor function. Despite the availability of dopamine-replacement strategies that generally offer considerable symptomatic relief early in the disease, as yet no therapy has been shown to slow the underlying neurodegenerative process. Adenosine A2A receptor antagonists recently have attracted attention as potential neuroprotective agents because of a remarkable convergence of epidemiological and laboratory data that link the A2A receptor to the development of PD (2Schwarzschild M.A. Chen J.-F. Ascherio A. Neurology. 2002; 58: 1154-1160Crossref PubMed Scopus (141) Google Scholar). Prospective studies of several large populations have shown that caffeine consumption is associated with a reduced risk of developing PD (3Ross G.W. Abbott R.D. Petrovitch H. Morens D.M. Grandinetti A. Tung K.H. Tanner C.M. Masaki K.H. Blanchette P.L. Curb J.D. Popper J.S. White L. JAMA. 2000; 283: 2674-2679Crossref PubMed Scopus (605) Google Scholar, 4Ascherio A. Zhang S.M. Hernan M.A. Kawachi I. Colditz G.A. Speizer F.E. Willett W.C. Ann. Neurol. 2001; 50: 56-63Crossref PubMed Scopus (553) Google Scholar). The risk of PD decreased with increasing prior intake of coffee or of caffeine from other sources and was independent of smoking status or other potential confounding factors. Notably, consumption of decaffeinated coffee was not related to PD risk (4Ascherio A. Zhang S.M. Hernan M.A. Kawachi I. Colditz G.A. Speizer F.E. Willett W.C. Ann. Neurol. 2001; 50: 56-63Crossref PubMed Scopus (553) Google Scholar). The possibility that the reduced risk of PD among caffeine consumers is due to a neuroprotective effect of caffeine has been supported by our finding that caffeine can reduce dopaminergic neuron toxicity in a mouse model of PD (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar). Low doses of caffeine can attenuate the loss of striatal dopamine and of dopamine transporter (DAT) binding sites induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The neuroprotection by caffeine, a nonspecific adenosine receptor antagonist (6Fredholm B.B. Bättig K. Holmén J. Nehlig A. Zvartau E.E. Pharmacol. Rev. 1999; 51: 83-133PubMed Google Scholar), could be mimicked by relatively specific adenosine A2A receptor antagonists but not an A1 antagonist (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar, 7Ikeda K. Kurokawa M. Aoyama S. Kuwana Y. J. Neurochem. 2002; 80: 262-270Crossref PubMed Scopus (198) Google Scholar). A2A receptor knockout mice also were resistant to MPTP-induced depletion of striatal dopamine. Together these laboratory data have suggested a potential neurobiological basis for the inverse association between caffeine use and PD. Here we examine the neuroprotective properties of 8-(3-chlorostyryl)caffeine (CSC), a selective and potent A2A antagonist closely related to caffeine (8Jacobson K.A. Nikodijevic O. Padgett W.L. Gallo-Rodriguez C. Maillard M. Daly J.W. FEBS Lett. 1993; 323: 141-144Crossref PubMed Scopus (163) Google Scholar) in the MPTP model of PD (9Gerlach M. Riederer P. J. Neural Transm. 1996; 103: 987-1041Crossref PubMed Scopus (437) Google Scholar). Because the neurotoxicity of MPTP requires its oxidation to the active toxin, the 1-methyl-4-phenylpyridinium (MPP+) species, by monoamine oxidase B (MAO-B), we investigated the effects of CSC on MPTP metabolism in vivoand on MAO activity in vitro. The results of these studies offer new insight into structure-activity relationships for MAO-B inhibitors and suggest a novel class of dual-function compounds with enhanced potential for the treatment of PD. Male C57Bl/6 mice (25–28 g; 2–3 months old) received a single intraperitoneal injection of 20–40 mg/kg MPTP·HCl (or saline) or four intraperitoneal injections of 20 mg/kg MPTP·HCl (or saline) two hours apart. Ten minutes prior to each MPTP dose mice were pretreated with CSC or vehicle (a fresh mixture of dimethyl sulfoxide (15%), ethoxylated castor oil (15%; Alkamuls EL-620, Rhodia, Cranberry, NJ), and water). A2A KO mice were generated using a standard displacement target vector as described previously (10Chen J.-F. Huang Z. Zhu J. Moratalla R. Stadaert D. Moskowitz M.A. Fink J.S. Schwarzschild M.A. J. Neurosci. 1999; 19: 192-200Google Scholar). Briefly, chimeric A2A KO mice (F0) derived from 129-Steel embryonic stem cells were bred to C57Bl/6 mice, resulting in mice of mixed C57Bl/6 × 129-Steel backgrounds. To effectively eliminate the potentially confounding influence of the 129-Steel background, the mixed line was then repeatedly back-crossed to pure C57Bl/6 mice over six generations yielding a near congenic (N6) C57Bl/6 line. A2A KO (−/−) and wild-type (WT, +/+) littermates (both male and female) from N6 heterozygote (±) intercrosses were used in this study. Seven days after treatment, mice were sacrificed by rapid cervical dislocation and assayed for striatal dopamine or serotonin content and [3H]mazindol (DAT) binding as described previously (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar). Striatal concentrations of MPTP, MPDP+, and MPP+ were measured as described previously (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar, 11Giovanni A. Sieber B.A. Heikkila R.E. Sonsalla P.K. J. Pharmacol. Exp. Ther. 1991; 257: 691-697PubMed Google Scholar). Intact mitochondria prepared from C57BL/6 mouse brain and human placenta served as sources of MAO. The mitochondrial fractions were prepared as described by Salach and Weyler (12Salach J. Weyler J. Methods Enzymol. 1987; 142: 627-637Crossref PubMed Scopus (59) Google Scholar) with minor modifications and were stored at −70 °C. Before use, the mitochondrial homogenate was suspended in sodium phosphate buffer (0.1 m, pH 7.4) containing 50% (w/v) glycerol. The protein concentrations (25–60 mg/ml) were determined by the method of Bradford (13Bradford M.M. Anal. Biochem. 1976; 72: 247-254Crossref Scopus (216178) Google Scholar). K m and V max determinations of the MAO-B-catalyzed oxidation of the MAO-B-selective substrate MPTP (25–400 μm) were carried out in brain mitochondrial preparations (final protein concentration of 0.3 mg protein/ml in 0.1 mm sodium phosphate buffer pH 7.4; 500 μl final volume; 30 min at 37 °C) obtained from WT and A2A KO mice (pooled tissues of three mice in each case; determinations in duplicate). The reactions were terminated by the addition of 20 μl of perchloric acid (70% v/v), and the samples were centrifuged at 16,000 × g for 5 min. The supernatant fractions were removed and assayed for the MAO-B-generated dihydropyridinium metabolite (MPDP+) by measuring the absorbance at 345 nm spectrophotometrically (ε = 16,000 m−1cm−1) (14Kalgutkar A. Castagnoli K. Hall A. Castagnoli Jr., N. J. Med. Chem. 1994; 37: 944-949Crossref PubMed Scopus (35) Google Scholar, 15Inoue H. Castagnoli K. Van der Schyf C. Mabic S. Igarashi K. Castagnoli Jr., N. J. Pharmacol. Exp. Ther. 1999; 291: 856-864PubMed Google Scholar). The activity ratios of MAO-A to MAO-B were determined in mouse brain mitochondrial preparations from A2A KO and WT mice (each ratio represents averaged duplicate values from three separate animals). To measure MAO-B activity, the MAO-A present in the mouse brain mitochondria was inactivated by preincubating the preparation (1.2 mg of protein/ml) with 3.3 × 10−8mclorgyline hydrochloride, an MAO-A-selective inhibitor, for 15 min at 37 °C in 0.1 m, pH 7.4 sodium phosphate buffer (15Inoue H. Castagnoli K. Van der Schyf C. Mabic S. Igarashi K. Castagnoli Jr., N. J. Pharmacol. Exp. Ther. 1999; 291: 856-864PubMed Google Scholar). This solution was added to an equal volume of a solution of the non-selective MAO-A/B substrate 1-methyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydroyridine (MMTP, final concentration of 2 mm) also in sodium phosphate buffer (500 μl final volume). Following a 30-min incubation period at 37 °C, the reactions were terminated by the addition of 20 μl of 70% perchloric acid. The resulting mixtures were centrifuged, and the concentrations of the enzyme-generated dihydropyridinium metabolite MMDP+ were measured spectrophotometrically at 420 nm (ε = 25 000 m−1 cm−1) (15Inoue H. Castagnoli K. Van der Schyf C. Mabic S. Igarashi K. Castagnoli Jr., N. J. Pharmacol. Exp. Ther. 1999; 291: 856-864PubMed Google Scholar). MAO-A activity was estimated in the same way, using mouse brain mitochondria pretreated with the MAO-B-selective inhibitor (R)-deprenyl (3.3 × 10−7m) (15Inoue H. Castagnoli K. Van der Schyf C. Mabic S. Igarashi K. Castagnoli Jr., N. J. Pharmacol. Exp. Ther. 1999; 291: 856-864PubMed Google Scholar). The total MAO activity was determined by carrying out this assay in the absence of inactivators. Studies on the inhibition of MAO-B by CSC, caffeine, 1,3-dipropyl-8-cyclopentylxanthine (CPX), and 3,7-dimethyl-1-propargylxanthine (DMPX) utilized the MAO-B-selective substrate MPTP (16Khalil A.A. Steyn S. Castagnoli Jr., N. Chem. Res. Toxicol. 2000; 13: 31-35Crossref PubMed Scopus (86) Google Scholar). The incubation mixtures (500 μl final volume in sodium phosphate buffer, pH 7.4) contained MPTP (30–90 μm), mouse brain mitochondrial homogenate (0.15 mg of protein/ml), and the appropriate concentrations of the compounds of interest. Caffeine was dissolved in sodium phosphate buffer. Because of limited water solubility, CSC, CPX, and DMPX were dissolved in 100% Me2SO and added to the buffered incubation mixtures such that the final Me2SO concentration was 4%. Previous studies in our laboratory have shown that solutions containing 4% Me2SO do not affect enzyme activity. The samples were incubated at 37 °C for 45 min, during which time the rate of oxidation of MPTP remained constant. The reactions were terminated by the addition of 20 μl of 70% perchloric acid, and the samples were centrifuged at 16,000 × g for 5 min. The supernatant fractions were removed and assayed for MPDP+ and MPP+ content using reverse phase high pressure liquid chromatography rather than spectrophotometrically because the CSC chromophore (λmax = 350 nm) overlapped with that of MPDP+ (λmax = 345 nm). The mobile phase consisted of 80% Milli-Q water (containing 0.6% (v/v) glacial acetic acid and 1% (v/v) triethylamine) and 20% acetonitrile at a flow rate of 1 ml/min. A volume of 200 μl of supernatant fraction was injected into the high pressure liquid chromatography system. MPDP+was monitored at 345 nm and MPP+ at 285 nm. Quantitative determinations of these metabolites were carried out with the aid of calibration curves that were prepared over the linear concentration ranges of interest (MPDP+, 0.8–3.0 μm; MPP+, 0.2–0.8 μm). These data were used to determine the initial velocity (V) of the MAO-B-catalyzed oxidation of MPTP. The double-reciprocal plots of 1/V (1/(rate of MPDP+ plus MPP+ formation)) versus1/(MPTP) with increasing concentrations of the inhibitor were constructed. The K i value (−x wheny = 0) was determined from a replot in which the values of the slopes obtained from these double reciprocal graphs were plotted against the concentration of the competitive inhibitor (x-axis) (17Segel I.H. Enzyme Kinetics. Wiley, New York1993: 100-125Google Scholar). Studies on the inhibition of MAO-A by CSC utilized the MAO-A/B non-selective substrate MMTP and human placental mitochondria, which express exclusively MAO-A (18Weyler W. Salach J. J. Biol. Chem. 1985; 260: 13199-13207Abstract Full Text PDF PubMed Google Scholar, 19Riley L.A. Waguespack M.A. Denney R.M. Mol. Pharmacol. 1989; 36: 54-60PubMed Google Scholar). Essentially the same protocol was followed as described above for the MAO-B inhibition studies with the exception that the incubation time was 15 min and the substrate concentrations ranged from 30 to 120 μm. The concentrations of the MAO-generated dihydropyridinium metabolite MMDP+ in the supernatant fractions were measured spectrophotometrically at a wavelength of 420 nm. K ivalues were determined as described above. Single statistical comparisons between two groups were performed using a non-paired two-tailed Student'st test. Analysis of dose-response relationships was performed by one-way analysis of variance followed by Dunnett'spost hoc comparisons. Data values present group averages ± S.E. The loss of striatal dopamine induced by MPTP (administered in four 20 mg/kg intraperitoneal doses two hours apart) in C57Bl/6 mice was significantly attenuated by CSC (5 mg/kg intraperitoneal 10 min prior to each MPTP dose; Fig.1 A, left panel). CSC also attenuated dopamine loss induced by a single high dose of MPTP·HCl (40 mg/kg), and it did so in a dose-dependent manner with complete protection observed at and above 20 mg/kg CSC (Fig. 1 B). In contrast to dopamine, serotonin levels in the striatum were not altered by MPTP (Fig. 1 A, right panel), highlighting the selectivity of the toxin for dopaminergic neurons. CSC had no effect on baseline levels of dopamine or serotonin in the striatum. In addition to a biochemical marker of nigrostriatal integrity (dopamine) DAT density in the striatum, an anatomical marker of nigrostriatal innervation, was also assessed. MPTP induced a loss of striatal DAT ([3H]mazindol) binding sites commensurate with that of striatal dopamine content. This loss was significantly attenuated by pretreatment with CSC (Fig. 1 C). Taken together with prior findings that mice pretreated with other specific A2A antagonists and those lacking functional A2A receptors showed reduced MPTP toxicity (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar, 7Ikeda K. Kurokawa M. Aoyama S. Kuwana Y. J. Neurochem. 2002; 80: 262-270Crossref PubMed Scopus (198) Google Scholar), these data seem to suggest that CSC protects dopaminergic neurons by blocking A2A receptors. Moreover, the locomotor stimulating effect of 5 mg/kg CSC was completely blocked in A2A receptor knockout mice, 2J.-F. Chen and M. A. Schwarzschild, unpublished observations. lending further support to the possibility that the neuroprotective effect of CSC at this dose depends on its A2A antagonist properties. Because the neurotoxicity of MPTP requires its oxidation to the active toxin MPP+, we examined the effects of CSC pretreatment on MPP+ levels in the striatum (Fig.2 A). Mice were treated with vehicle or CSC 5 min prior to each of the four MPTP injections. 90 min after the last MPTP injection, striatal MPP+ levels were significantly lower in CSC-treated mice compared with those treated with vehicle. Thus CSC leads to decreased MPP+ levels in the striatum, which may contribute to its attenuation of MPTP toxicity. To investigate further the potential mechanism underlying attenuated MPP+ levels in the striatum, we also determined the effects of CSC on striatal levels of MPTP and MPDP+ following intraperitoneal MPTP treatment. After crossing the blood-brain barrier, MPTP is oxidized in a reaction catalyzed by MAO-B to yield the relatively unstable 1-methyl-4-phenyl-2,3-dihydropyridinium intermediate MPDP+, which in turn oxidizes further to the stable active toxin MPP+ (9Gerlach M. Riederer P. J. Neural Transm. 1996; 103: 987-1041Crossref PubMed Scopus (437) Google Scholar). 15 min after a single intraperitoneal injection of MPTP, striatal levels of MPTP and MPDP+ peak while striatal MPP+ levels are starting to rise (11Giovanni A. Sieber B.A. Heikkila R.E. Sonsalla P.K. J. Pharmacol. Exp. Ther. 1991; 257: 691-697PubMed Google Scholar). Pretreatment with CSC (5 mg/kg) significantly increased MPTP levels and decreased both MPDP+ and MPP+ levels in striatum at 15 min post-MPTP administration (Fig. 2 B), an effect also seen with the MAO-B inhibitor, 7-nitroindazole (27Castagnoli K. Palmer S. Anderson A. Bueters T. Castagnoli Jr., N. Chem. Res. Toxicol. 1997; 10: 1771-1773Google Scholar). To explore the in vivo metabolism of MPTP in the more complex (but pathophysiologically more relevant) multiple-dose toxin paradigm, we quantified striatal metabolites 15 min after the four injections of MPTP·HCl (20 mg/kg × 4, intraperitoneal, Fig.2 C). In this case striatal levels of MPTP and MPDP+ reflect principally the fate of the last dose of MPTP, whereas the level of MPP+ reflects the cumulative effects of the three prior injections (11Giovanni A. Sieber B.A. Heikkila R.E. Sonsalla P.K. J. Pharmacol. Exp. Ther. 1991; 257: 691-697PubMed Google Scholar). As in the single injection study, 15 min after the fourth MPTP injection increased levels of MPTP and decreased levels of MPDP+ and MPP+ were observed in mice pretreated with CSC (5 mg/kg, before each MPTP dose) compared with mice pretreated with vehicle. Together these in vivo MPTP metabolite data indicate that CSC does not attenuate MPTP delivery to striatum; rather, it appears to attenuate striatal conversion of MPTP to MPDP+ and MPP+. By contrast, MPTP and MPDP+ levels are unaltered in the striatum of A2A KO compared with wild-type mice and in mice pretreated with the nonspecific adenosine antagonist caffeine compared with vehicle (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar). The inhibition of MPTP metabolismin vivo by CSC but not by certain other antagonists of A2A receptors or by A2A receptor deficiency (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar,7Ikeda K. Kurokawa M. Aoyama S. Kuwana Y. J. Neurochem. 2002; 80: 262-270Crossref PubMed Scopus (198) Google Scholar) suggests that A2A receptors do not regulate MAO-B activity and thus raises the possibility that CSC may act as an MAO-B inhibitor independent of its A2A antagonist properties. To investigate the possibility of a direct effect of CSC on MAO activity, we assayed mitochondrial MAO-A and MAO-B activities in the presence of CSC across a range of concentrations. Fig. 3 shows that CSC potently and competitively inhibits MAO-B activity in a mitochondrial preparation from mouse brain with a K ivalue of ∼100 nm, a value comparable with that of the most potent known competitive MAO-B inhibitors (20Krueger M.J. Mazoua F. Ramsay R.R. Milcent R. Singer T.P. Biochem. Biophys. Res. Comm. 1995; 206: 556-562Crossref PubMed Scopus (53) Google Scholar). In contrast to MAO-B, MAO-A (from human placenta) was not significantly inhibited by CSC. To determine whether other adenosine receptor antagonists also share this unexpected property of MAO-B inhibition we compared the effect of CSC with those of caffeine (a nonspecific adenosine receptor antagonist), DMPX (another relatively specific adenosine A2A receptor antagonist), and CPX (a relatively specific adenosine A1 receptor antagonist) on MAO-B activity. In studies using a mitochondrial preparation from mouse brain with MPTP as substrate, CSC potently inhibited MAO-B activity in vitrowith a K i of ∼100 nm, whereas caffeine, DMPX, and CPX produced little if any inhibition with estimated K i values of 0.7, 1, and ≥4 mm, respectively. These findings demonstrate that, in addition to its A2A antagonist properties, CSC also is a potent and selective inhibitor of MAO-B. Our observation that 1 μm CSC completely blocks MAO-B activity in primary cultures of brain glia, which express few if any A2Areceptors, further suggests the possibility that CSC inhibits MAO-B directly. 3K. Xu, J.-F. Chen, and M. A. Schwarzschild, unpublished data. Although the MAOs are not known to couple to receptors, the standard mitochondrial preparations used to identify the MAO inhibitory properties of CSC are likely to contain A2Areceptors through which CSC could indirectly inhibit MAO-B. The recent demonstration of an ultrastructural localization of A2Areceptors to intracellular organelle membranes within striatal neurons (21Hettinger B.D. Lee A. Linden J. Rosin D.L. J. Comp. Neurol. 2001; 431: 331-346Crossref PubMed Scopus (234) Google Scholar) underscores the need to address this possibility. To assess A2A receptor involvement in the effect of CSC on MAO-B, we took advantage of an A2A KO model of A2Areceptor function (10Chen J.-F. Huang Z. Zhu J. Moratalla R. Stadaert D. Moskowitz M.A. Fink J.S. Schwarzschild M.A. J. Neurosci. 1999; 19: 192-200Google Scholar). We first compared the MAO activities in the brains of A2A KO and WT mice using MMTP as substrate (TableI). Under V maxconditions in the presence of appropriate inhibitors, no significant difference was observed in the total MAO activity or the activities of MAO-A and MAO-B. In a separate experiment the K m andV max values of MAO-B-catalyzed oxidation of MPTP also were found to be indistinguishable in brain mitochondrial preparation from the A2A KO mice and WT littermates (TableI). The normal kinetics of MAO activity in A2A KO (as well as the absence of MAO inhibitory activity of the A2Aantagonist DMPX) argue against a modulatory effect of A2Areceptors on MAO-B activity.Table IComparing MAO-B activities in brain mitochondrial preparations from WT and A2A KO miceWTA2A KOt-testMAO activities1-anmol MMDP+ formed/min-mg mitochondrial protein.Total2.23 ± 0.151.95 ± 0.08p>0.1MAO-B1.68 ± 0.111.49 ± 0.09p>0.2MAO-A0.48 ± 0.050.41 ± 0.02p>0.2Michaelis-Menten parametersK m66.11 ± 2.271-bμM.66.64 ± 0.15p>0.8V max0.88 ± 0.021-cnmol MPDP+ metabolite formed/min-mg mitochondrial protein.0.87 ± 0.00p>0.6V max/K m13.27 ± 0.2113.01 ± 0.03p>0.31-a nmol MMDP+ formed/min-mg mitochondrial protein.1-b μM.1-c nmol MPDP+ metabolite formed/min-mg mitochondrial protein. Open table in a new tab We also examined the inhibitory effects of CSC on MAO-B activity of mitochondria prepared from the brains of A2A KO mice and their WT littermates. Fig. 4 shows that CSC is just as potent in its inhibition of A2A KO MAO-B as it is in its inhibition of WT MAO-B (with a K i of ∼100 nm for each). These data confirm the hypothesis that the novel MAO-B inhibitory action of CSC is independent of its well established antagonistic action on A2A receptors. A direct inhibition of MAO-B explains the reduction in the levels of MPDP+ and the active toxin MPP+ in vivo when systemic MPTP is administered with CSC (Fig. 2) but not with other A2A antagonists (5Chen J.-F., Xu, K. Petzer J.P. Staal R., Xu, Y.-H. Beilstein M. Sonsalla P.K. Castagnoli K. Castagnoli Jr., N. Schwarzschild M.A. J. Neurosci. 2001; 21: 1-6PubMed Google Scholar, 7Ikeda K. Kurokawa M. Aoyama S. Kuwana Y. J. Neurochem. 2002; 80: 262-270Crossref PubMed Scopus (198) Google Scholar). Thus, the neuroprotective effect of CSC in the MPTP model of PD may rely in part on this A2A receptor-independent inhibition of MAO-B. The unexpected finding of dual MAO-B inhibitory and A2Areceptor antagonistic function in a xanthine-derived structure may offer valuable biological and pharmacological insights and opportunities. The recently reported x-ray structure of MAO-B (28Binda C. Newton-Vinson P. Hubalik F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2001; 9: 22-26Crossref Scopus (532) Google Scholar) together with further structure-function relationship studies now underway should help to identify possible relationships between the active sites of these two proteins. The pharmacological significance of a single structure capable of both MAO-B inhibition and A2A receptor antagonism is underscored by ongoing clinical trials that are based on these two individual anti-parkinsonian strategies. Moreover, the targeting of either of these proteins may be particularly beneficial in treating PD because both MAO-B inhibitors and A2A antagonists possess neuroprotective as well as symptomatic therapeutic potential (22Kanthasamy, A. G., Sharma, N., Kirby, M. L., and Schwarzschild, M. A. in Neuroprotection (Marwah, J., and Lo, E., eds) pp. 603–640, Prominent Press, Scottsdale, AZ.Google Scholar). The neuroprotective benefits of dual-function agents offering MAO inhibition and A2A antagonism may extend beyond PD because preclinical studies have suggested possible therapeutic effects of both MAO inhibitors and A2A antagonists in a range of neuropsychiatric disorders from stroke to depression (10Chen J.-F. Huang Z. Zhu J. Moratalla R. Stadaert D. Moskowitz M.A. Fink J.S. Schwarzschild M.A. J. Neurosci. 1999; 19: 192-200Google Scholar, 23Yu P.H. Davis B.A. Zhang X. Zuo D.M. Fang J. Lai C.T., Li, X.M. Paterson I.A. Boulton A.A. Prog. Brain Res. 1995; 106: 113-121Crossref PubMed Scopus (22) Google Scholar, 24El Yacoubi M. Ledent C. Parmentier M. Bertorelli R. Ongini E. Costentin J. Vaugeois J.-M. Br. J. Pharmacol. 2001; 134: 68-77Crossref PubMed Scopus (168) Google Scholar). The recognition that CSC acts as an MAO-B inhibitor as well as an A2A antagonist also may help to explain an unexpected observation on the brain distribution of isotopically labeled CSC (25Marian T. Boros I. Lengyel Z. Balkay L. Horvath G. Emri M. Sarkadi E. Szentmiklosi A.J. Fekete I. Tron L. Appl. Radiat. Isot. 1999; 50: 887-893Crossref PubMed Scopus (26) Google Scholar). This compound, which was designed as a positron emission tomography ligand for measuring A2A receptor density in humans, was found to label most heavily the relatively A2Areceptor-poor region of the ventral medulla in addition to the A2A receptor-rich striatum. That the ventral medulla contains a high density of serotonergic neurons known to express high levels of MAO-B (26Kitahama K. Denney R.M. Maeda T. Jouvet M. Neuroscience. 1991; 44: 185-204Crossref PubMed Scopus (29) Google Scholar) fits well with the present finding that CSC acts on MAO-B as well as the A2A receptor. In conclusion, the present data indicate that CSC possesses dual actions of MAO-B inhibition and A2A receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced therapeutic potential in PD and other neuropsychiatric disorders.
Previous studies have established that the tobacco alkaloid 1-methyl-2-(3-pyridyl)pyrrole (β-nicotyrine) is biotransformed by rabbit lung and liver microsomal preparations to an equilibrium mixture of the corresponding 3- and 4-pyrrolin-2-ones. Autoxidation of these pyrrolin-2-ones generates the chemically stable 5-hydroxy-5-(3-pyridinyl)-3-pyrrolin-2-one. This paper summarizes efforts to document more completely the pathway leading to this hydroxypyrrolinone. Chemical and spectroscopic evidence implicates the 2-hydroxy-1-methyl-5-(3-pyridinyl)pyrrole (2-hydroxy-β-nicotyrine) as the key intermediate in this reaction pathway. Of potential toxicological interest is the detection of radical species derived from the autoxidation of this compound.
In order to gain more insight into the mechanisms underlying mitochondrial inhibition by haloperidol (HP) pyridinium metabolites, we have studied the three dimensional structure of these compounds. In this paper we report the results of experimental (NMR studies in solution, X-ray diffraction) and theoretical methods (molecular dynamics) applied to HPP+. The chlorophenyl and pyridinium rings are found not to be strictly coplanar and a high degree of mobility was observed in the butyroxy chain. Calculations have shown that the most stable structures adopt conformations corresponding to either gauche or trans rotamers. From these data, a model for the interaction of HPP+ with electron transfer complexes in the mitochondrial respiratory chain has been proposed.
Abstract Polycyclic cage scaffolds have been successfully used in the development of numerous lead compounds demonstrating activity in the central nervous system (CNS). Several neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, schizophrenia, and stroke, as well as drug abuse, can be modulated with polycyclic cage derivatives. These cage moieties, including adamantane and pentacycloundecane derivatives, improve the pharmacokinetic and pharmacodynamic properties of conjugated parent drugs and serve as an important scaffold in the design of therapeutically active agents for the treatment of neurological disorders. In this Minireview, we focus on the recent developments in the field of polycyclic cage compounds, as well as the relationship between the lipophilic character of these cage‐derived drugs and the ability of such compounds to target and reach the CNS and improve the pharmacodynamic properties of compounds conjugated to it.
In the crystal structure of (E)-8-(3-chlorostyryl)-1,3,7-trimethylxanthine (CSC) [systematic name: (E)-8-(3-chlorostyryl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione], C16H15ClN4O2, the xanthine ring and the lateral styryl chain are coplanar. The crystal packing involves mainly parallel stacking of these planar molecules. The electrostatic potential calculated on the crystal structure conformation confirms the pharmacophore elements associated with MAO-B inhibition.
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