Document Information

PMID 16188953  (  )
Title Targeted antioxidative and neuroprotective properties of the dopamine agonist pramipexole and its nondopaminergic enantiomer SND919CL2x [(+)2-amino-4,5,6,7-tetrahydro-6-Lpropylamino-benzathiazole dihydrochloride].
Abstract Pramipexole has been shown to possess neuroprotective properties in vitro that are partly independent of its dopaminergic agonism. The site of neuroprotective action is still unknown. Using [(3)H]pramipexole, we show that the drug enters and accumulates in cells and mitochondria. Detoxification of reactive oxygen species (ROS) by pramipexole is shown in vitro and in vivo by evaluating mitochondrial ROS release and aconitase-2 activity, respectively. Pramipexole and its (+)-enantiomer SND919CL2X [low-affinity dopamine agonist; (+)2-amino-4,5,6,7-tetrahydro-6-l-propylamino-benzathiazole dihydrochloride] possess equipotent efficacy toward hydrogen peroxide and nitric oxide generated in vitro and inhibit cell death in glutathione-depleted neuroblastoma cells. IC(50) values ranged from 15 to 1000 microM, consistent with the reactivity of the respective radical and the compartmentalization of ROS generation and ROS detoxification. Finally, both compounds were tested in superoxide dismutase 1-G93A mice, a model of familial amyotrophic lateral sclerosis. SND919CL2X (100 mg/kg) prolongs survival time and preserves motor function in contrast to pramipexole (3 mg/kg), which shows an increase in running wheel activity before disease onset, presumably caused by the dopaminergic agonism. We conclude that both enantiomers, in addition to their dopaminergic activity, are able to confer neuroprotective effects by their ability to accumulate in brain, cells, and mitochondria where they detoxify ROS. However, a clinical use of pramipexole as a mitochondria-targeted antioxidant is unlikely, because the high doses needed for antioxidative action in vitro are not accessible in vivo due to dopaminergic side effects. In contrast, SND919CL2X may represent the prototype of a mitochondria-targeted neuroprotectant because it has the same antioxidative properties without causing adverse effects. GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany. lothar.kussmaul@bc.boehringer-ingelheim.com.

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Targets by SciMiner Summary

HUGO ID Symbol Target Name #Occur ActualStr
118ACO2aconitase 2, mitochondrial14aconitase | aconitase 2 | aconitase 2 mitochondrial |
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))12superoxide dismutase 1 | SOD-catalase | SOD1 | SOD-catalase-mimetic |
12805XDHxanthine dehydrogenase6xanthine oxidase |
1516CATcatalase3catalase |
5385IDH3Bisocitrate dehydrogenase 3 (NAD+) beta1isocitrate dehydrogenase |
17348PRPF3PRP3 pre-mRNA processing factor 3 homolog (S. cerevisiae)1RP18 |

 

Targets by SciMiner Full list

HUGO ID Symbol Name ActualStr Score FlankingText
118ACO2aconitase 2, mitochondrialaconitase1.62 O 2 release and by an increase in mitochondrial aconitase activity
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD12.2cases are caused by mutations in superoxide dismutase 1 (SOD1), SOD1 which expressed in mice result in a phenotype resembling the
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD12.2Transgenic SOD1 mice represent the predominant model to study ALS pathogenesis and
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD12.2test for neuroprotection by PPX/SND PPX SND in vivo transgenic SOD1 mice were treated with both compounds
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD-catalase2.2PPX (300 300 microM malonate (Malo, Malo 10 mM the SOD-catalase mimic EUK-134 (Melov Melov et al. 2001 EUK (30 30
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD2.2The specificity for hydrogen peroxide was verified with SOD (300 300 mU/ml), mU ml which did not affect the
17348PRPF3PRP3 pre-mRNA processing factor 3 homolog (S. cerevisiae)RP181.0Chromatography took place on a Kromasil RP18 column (2.1 2.1 mm i.d x 30 mm 5-microm particle
118ACO2aconitase 2, mitochondrialaconitase1.6Mitochondrial aconitase is reversibly inactivated by superoxide and has therefore been used
118ACO2aconitase 2, mitochondrialaconitase1.6against the 23 C-terminal amino acids of aconitase-2 (mitochondrial mitochondrial aconitase from rat (nanoTools; nanoTools Antik_amp_ouml rpertechnik Teningen Germany was used
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD-catalase-mimetic2.2and compartments obtained efficacies were compared with EUK-134 a potent SOD-catalase-mimetic (Jung Jung et al. 2001 Melov et al. 2001
118ACO2aconitase 2, mitochondrialaconitase1.630 mg/kg) mg kg results in an increase in mitochondrial aconitase activity in vivo indicating a reduction of the superoxide steady-state
118ACO2aconitase 2, mitochondrialaconitase1.612 h after the last treatment the increase in mitochondrial aconitase might result from total brain concentrations within the same order
118ACO2aconitase 2, mitochondrialaconitase1.6PPX causes a slight but not significant increase in total aconitase activity in 1-methyl-4-phenylpyridinium-treated SHSY-5Y cells
118ACO2aconitase 2, mitochondrialaconitase1.6specific endpoints such as modification of mitochondrial proteins or mitochondrial aconitase activity
118ACO2aconitase 2, mitochondrialaconitase1.6Activity (A) A and expression (B) B of mitochondrial aconitase after treatment of C57BL/6 C57BL 6 mice with PPX (2
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD12.2Treatment of SOD1(G93A) SOD1 G93A transgenic mice with PPX and SND
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD-catalase2.2SND and compared the obtained efficacy with EUK-134 a potent SOD-catalase mimetic (Melov Melov et al. 2001
118ACO2aconitase 2, mitochondrialaconitase1.6were isolated after PPX treatment of mice to determine mitochondrial aconitase activity an indicator for steady-state levels of superoxide (Tarpey Tarpey
118ACO2aconitase 2, mitochondrialaconitase1.6days (2 2 x 30 mg/kg) mg kg increased mitochondrial aconitase activity by 42% to 67 _amp_#177 16 mU/mg mU mg
118ACO2aconitase 2, mitochondrialaconitase1.6This was not due to altered expression of mitochondrial aconitase because protein levels were not affected by the PPX treatment
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD12.2Consequently we tested both compounds in mice expressing the G93A-mutant SOD1 a common and well described model of ALS where neuronal
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD12.2Additionally survival time in SND-treated SOD1 (G93A) G93A mice (132 132 _amp_#177 2 days mean _amp_#177
118ACO2aconitase 2, mitochondrialaconitase 21.0detoxification of reactive oxygen species ros by pramipexole is shown in vitro and in vivo by evaluating mitochondrial ros release and aconitase 2 activity respectively.
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))superoxide dismutase 11.0finally both compounds were tested in superoxide dismutase 1 g93a mice a model of familial amyotrophic lateral sclerosis.
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))superoxide dismutase 11.0twenty percent of familial als cases are caused by mutations in superoxide dismutase 1 sod1 which expressed in mice result in a phenotype resembling the pathology in patients.
1516CATcatalasecatalase1.0in further conditions ppx 300 microm malonate malo 10 mm the sod catalase mimic euk 134 melov et al. 2001 euk 30 microm or atp 1 mm was added.
12805XDHxanthine dehydrogenasexanthine oxidase1.0in a further set of experiments hydrogen peroxide ~80% and to a smaller extent superoxide ~20% were generated by xanthine oxidase 0.3 mu/ml and hypoxanthine 0.1 mm fridovich 1970 in a solution containing ppx snd or euk 134 at the indicated concentrations.
5385IDH3Bisocitrate dehydrogenase 3 (NAD+) betaisocitrate dehydrogenase1.0twenty microliters of lysate was added to a well of a 96 well microtiter plate followed by the addition of a prewarmed solution 37degreec containing 0.4 mm nadp 1.2 mm mncl 2 and 2 u/ml isocitrate dehydrogenase in buffer [10 mm tris/hcl 0.6 mm mncl 2 20 microm d l fluorocitrate and 1% v/v triton x 100].
118ACO2aconitase 2, mitochondrialaconitase 2 mitochondrial1.0additionally a monoclonal antibody raised against the 23 c terminal amino acids of aconitase 2 mitochondrial aconitase from rat nanotools; antik_amp_ouml;rpertechnik teningen germany was used to study the expression of the protein in mitochondria.
118ACO2aconitase 2, mitochondrialaconitase 21.0afterward aconitase 2 expression was analyzed by a standard western blot protocol by probing the used nitrocellulose membranes with the anti aconitase 2 antibody 2 microg/ml using an enhanced chemiluminescence detection system applied biosystems .
1516CATcatalasecatalase1.0therefore we reevaluated the antioxidative properties of ppx and snd targeted toward different reactive species and compartments; obtained efficacies were compared with euk 134 a potent sod catalase mimetic jung et al. 2001 ; melov et al. 2001 .
12805XDHxanthine dehydrogenasexanthine oxidase1.0we show that ppx and the equipotent snd are weak h 2 o 2 scavengers if generation and detoxification are measured in the same compartment in absence of a detection system xanthine oxidase .
12805XDHxanthine dehydrogenasexanthine oxidase1.0a ros were generated by xanthine oxidase in a solution containing euk 134 black bars ppx light gray bars or snd dark gray bars at the indicated concentrations.
1516CATcatalasecatalase1.0after demonstrating entry of ppx into brain cells and mitochondria we reevaluated the antioxidative properties of ppx and snd and compared the obtained efficacy with euk 134 a potent sod catalase mimetic melov et al. 2001 .
12805XDHxanthine dehydrogenasexanthine oxidase1.0first h 2 o 2 and superoxide were generated by xanthine oxidase fridovich 1970 in a cell free assay where detoxifying enzymes and membranes are absent.
12805XDHxanthine dehydrogenasexanthine oxidase1.0as expected euk 134 was the most potent compound which at a concentration of 300 microm was able to detoxify >95% ros generated by xanthine oxidase.
12805XDHxanthine dehydrogenasexanthine oxidase1.0additionally both enantiomers showed equipotent efficacy in detoxification of xanthine oxidase generated ros fig 3a .