Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Localization of N-methyl-norsalsolinol within rodent and human brain.

The isoquinoline derivative N-methyl-6,7-dihydroxytetrahydroisoquinoline (N-methyl-norsalsolinol) is present in normal human brain and has been identified in the cerebrospinal fluid of patients with Parkinson's disease (PD). Endogenously, N-methyl-norsalsolinol may be derived from dopamine by condensation with aldehydes or alpha-ketoacids. In vitro experiments suggest that N-methyl-norsalsolinol is neurotoxic. In this study, high-performance liquid chromatography with electrochemical detection (HPLC-EC) was used to determine N-methyl-norsalsolinol concentrations in mouse, rat, normal human, and PD brain. In addition, a monoclonal antibody was generated against N-methyl-norsalsolinol and used to determine the cellular localization of N-methyl-norsalsolinol in brain. With HPLC-EC, N-methyl-norsalsolinol was detected in all regions of rodent and human brain subjected to analysis. In rodent brains, N-methyl-norsalsolinol tissue concentrations were similar among frontal cortex, ventral midbrain, striatum, hippocampus, and cerebellum. Conversely, in normal human control brains, N-methyl-norsalsolinol was concentrated in the substantia nigra and striatum. In comparison to normal human controls, N-methyl-norsalsolinol levels were significantly lower in the substantia nigra and caudate nuclei from PD patients, a finding possibly related to the death of nigrostriatal dopaminergic neurons. N-methyl-norsalsolinol immunoreactivity colocalized with a general neuronal marker (neuron-specific enolase) and a monoaminergic marker (tyrosine hydroxylase) but not with a glial marker (glial fibrillary acidic protein). The widespread neuronal localization of N-methyl-norsalsolinol in several mammalian species suggests that, in isolation, this compound is a "weak" neurotoxin. However, endogeneously derived N-methyl-norsalsolinol could contribute to the pathobiology of PD in genetically predisposed individuals after years of accumulation in dopaminergic neurons.

Cell death of dopamine neurons in aging and Parkinson's disease.

Dopamine neurons in the substantia nigra of human brain are selectively vulnerable and the number decline by aging at 5-10% per decade. Enzymatic and non-enzymatic oxidation of dopamine generates reactive oxygen species, which induces apoptotic cell death in dopamine neurons. Parkinson's disease (PD) is also caused by selective cell death of dopamine neurons in this brain region. The pathogenesis of Parkinson's disease remains to be an enigma, but it was found that an endogenous MPTP-like neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl(R)salsolinol, NM(R)Sal], may be one of the pathogenic agents of PD. NM(R)Sal increases in cerebrospinal fluid from untreated parkinsonian patients, and two enzymes, a (R)salsolinol synthase and a neutral N-methyltransferase, synthesize this neurotoxin in the nigro-striatum. The activity of a neutral N-methyltransferase is significantly higher in lymphocytes from parkinsonian patients than in control. The mechanism of cell death by this toxin was proved to be by the induction of apoptosis, by use of dopaminergic SH-SY5Y cells. The apoptosis was suppressed by anti-oxidants, suggesting that the generation of reactive oxygen species may initiate cellular death process. These results indicate that in aging and PD oxidative stress induces degeneration of dopamine neurons, and the antioxidant therapy may delay the decline of dopamine neurons in the brain.

Cell death in Parkinson's disease.

The cause of neuronal cell death in Parkinson's disease is still an enigma. However, recent results obtained by analyses of postmortem brain suggest that a mitochondria-dependent apoptotic signal was activated. The involvement of dopamine-derived endogenous neurotoxin in the pathogenesis of PD was also indicated. N-Methyl( R)salsolinol was proved to be selectively toxic to dopamine neurons and its level increased in parkinsonian CSF. The enzyme which determines the level of N-methyl( R)salsolinol, ( R)salsolinol N-methyltransferase, was found increased in the lymphocytes prepared from PD patients. The mechanism of dopamine cell death by N-methyl( R)salsolinol was studied in vitro. N-Methyl( R)salsolinol induced apoptosis in human dopaminergic neuroblastoma cells. It was suggested that in the mitochondria there is a molecule which interacts with N-methyl( R)salsolinol and initiates an apoptotic signal.

A dopaminergic neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl(R)salsolinol, and its oxidation product, 1,2(N)-dimethyl-6,7-dihydroxyisoquinolinium ion, accumulate in the nigro-striatal system of the human brain.

N-Methyl(R)salsolinol was found to be an endogenous dopaminergic neurotoxin inducing parkinsonism in rodents and to increase in the cerebrospinal fluid of parkinsonian patients. The amounts of N-methyl(R)salsolinol and related compounds in the human brain regions were quantitatively analyzed. Only the (R)-enantiomer of salsolinol derivatives were detected, which suggests their enzymatic synthesis in situ. In the nigro-striatal system, the concentration of N-methyl(+)salsolinol was higher than in the frontal cortex, and its oxidized catechol isoquinolinium ion was detected only in the substantia nigra significantly. The accumulation of these neurotoxins in the nigro-striatal region might account for selective cell death of dopamine neurons in the substantia nigra of Parkinson's disease.

Involvement of endogenous N-methyl(R)salsolinol in Parkinson's disease: induction of apoptosis and protection by (-)deprenyl.

An endogenous dopamine-derived N-methyl(R)salsolinol has been suggested to be involved in the pathogenesis of Parkinson's disease. In Parkinson's disease, the level of N-methyl(R)salsolinol increased in cerebrospinal fluid and the high activity of a synthesizing enzyme, (R)salsolinol N-methyltransferase, was detected in lymphocytes. This isoquinoline induced apoptotic DNA damage in human dopaminergic neuroblastoma SH-SY5Y cells. Among catechol isoquinolines, only N-methylsalsolinol induced apoptosis in the cells, and the scavengers of hydroxyl radicals and antioxidants suppressed DNA damage, suggesting that reactive oxygen species initiate apoptosis. The isoquinoline activated caspase-3 like proteases and a caspase-3 inhibitor protected the cells from DNA damage. (-)Deprenyl, but neither clorgyline nor pargyline, prevented apoptotic cell death. The mechanism of the protection was due to stabilization of mitochondrial membrane potential reduced by the toxin. In Parkinson's disease apoptosis may be induced in dopamine neurons by this endogenous neurotoxin, and (-)deprenyl may protect them from apoptotic death process.

N-methyl-(R)salsolinol as a dopaminergic neurotoxin: from an animal model to an early marker of Parkinson's disease.

A dopamine-derived 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydrosioquinoline [N-methyl-(R)salsolinol] was found to occur enantioselectively in human brain. This isoquinoline induced parkinsonism in rat after injection in the striatum, and the behavioral, biochemical and pathological changes were very similar to those in Parkinson's disease. N-Methyl-(R)salsolinol depleted dopamine neurons in the rat substantia nigra without necrotic tissue reaction, which may be due to the apoptotic death process, as proved by its induction of DNA damage in dopaminergic neuroblastoma SH-SY5Y cells. N-Methyl-(R)salsolinol was found to increase significantly in the cerebrospinal fluid of parkinsonian patients. All these results suggest that N-methyl-(R)salsolinol may be an endogenous neurotoxin to cause Parkinson's disease and the enzymes involved in its biosynthesis and catabolism may be endogenous factors in the pathogenesis of this disease.

Dopamine-derived endogenous N-methyl-(R)-salsolinol: its role in Parkinson's disease.

A dopamine-derived alkaloid, N-methyl-(R)-salsolinol [NM(R)Sal], enantioselectively occurs in human brains and accumulates in the nigrostriatal system. It increases in the cerebrospinal fluid (CSF) of parkinsonian patients and the activity of a neutral (R)-salsolinol [(R)Sal] N-methyltransferase, a key enzyme in the biosynthesis of this toxin, increases in the lymphocytes from parkinsonian patients, suggesting its involvement in the pathogenesis of Parkinson's disease (PD). The studies of animal and cellular models of PD proved that this isoquinoline is selectively cytotoxic to dopamine neurons. Using human dopaminergic SH-SY5Y cells, NM(R)Sal induces apoptosis by the activation of the apoptotic cascade initiated in mitochondria. In this article, we review the recent advance in proving our hypothesis that the dopamine-derived neurotoxin causes the selective depletion of dopamine neurons in PD.

Effect of ethanol on (R)- and (S)-salsolinol, salsoline, and THP in the nucleus accumbens of AA and ANA rats.

Tetrahydroisoquinolines (TIQ) are active metabolites of dopamine. Intracerebral application stimulates the voluntary ethanol intake. In the present study, the levels of several TIQ's [(S)- and (R)-salsolinol, salsoline and tetrahydropapaveroline (THP)] were measured in the extracellular space of the nucleus accumbens of alcohol-preferring AA and alcohol-avoiding ANA rats. Ethanol (2 g/kg i.p.) caused an increase in dopamine levels in ANA but not in AA rats. Neither (R)- nor (S)-salsolinol concentrations changed after ethanol application, though (S)-salsolinol concentrations were higher in ANA than in AA rats. Ethanol caused an increase in salsoline concentrations in ANA but not in AA rats. THP increased following ethanol, which tended to be stronger in ANA rats. The study revealed differences in the TIQ levels of the nucleus accumbens between AA and ANA rats. In case of changes following ethanol application (dopamine, salsoline, THP), the AA rats were less sensitive. The findings resemble observations in high-risk sons of alcoholics with reduced sensitivity to ethanol in young age and increased risk to become alcoholic.

A systematic regional study of dopamine and dopamine-derived salsolinol and norsalsolinol levels in human brain areas.

Dopamine and the dopamine-derived tetrahydroisoquinoline alkaloids salsolinol and norsalsolinol were measured by high-performance liquid chromatography with electrochemical detection in 15 regions of the human brain. The regional distribution of dopamine in 32 brains was similar to previous reports with highest concentrations in the basal ganglia, especially in the striatum, followed by the substantia nigra and the hypothalamus. Significant amounts of salsolinol and norsalsolinol were only found in these dopamine-rich areas, whereas in the other regions no alkaloids were detected. These findings suggest that the concentration of the substrate dopamine may determine the alkaloid level during in vivo formation.

Determination of dopamine and dopamine-derived (R)-/(S)-salsolinol and norsalsolinol in various human brain areas using solid-phase extraction and gas chromatography/mass spectrometry.

Using a solid-phase extraction procedure and a gas chromatographic-mass spectrometric (GC/MS) method the levels of dopamine and the levels of dopamine-derived salsolinol (SAL) and norsalsolinol (NorSAL) were determined in human brain areas involved in the etiology of alcoholism, parkinsonism and other diseases. The possibility that biosynthesis of salsolinol occurs through a stereospecific enzymatic reaction was considered. Using a two-step derivatization with N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA) and the chiral reagent (R)-(-)-2-phenylbutyryl chloride, baseline separated peaks of (R)- and (S)-SAL were obtained. Both enantiomers were found in human brain samples with no correlations between levels of salsolinol and dopamine. These findings do not support the hypothesis that only an enantio-selective synthesis of (R)-SAL by a putative salsolinol synthase is responsible for the in vivo formation. In our opinion, non-enzymatic formation of salsolinol via the Pictet-Spengler reaction reveals both salsolinol enantiomers and an additional enzymatic synthesis of only (R)-SAL explains the enantiomer ratio (R)-/(S)-SAL of approximately 2.

[Pathogenesis of idiopathic Parkinson's disease].

The pathogenesis of idiopathic Parkinson's disease (PD) remains to be elucidated. The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) suggests that neurotoxins in the human brain may cause selective depletion of striatal dopamine neurons, a hallmark of PD. An endogenous isoquinoline, N-methyl(R)salsolinol is a most promising neurotoxin candidate, and it was proved to be selectively toxic to dopamine neurons in the rat brain by in vivo experiments. The level of N-methyl(R)salsolinol in the cerebrospinal fluid obtained from PD patients was significantly higher than control. N-Methyl(R)salsolinol is synthesized by 2 enzymatic reactions from dopamine; condensation of dopamine with acetaldehyde into (R)salsolinol by (R)salsolinol synthase and N-methylation of (R)salsolinol by neutral(R)salsolinol N-methyltransferase. The second enzyme, which catabolizes the N-methylation of (R)salsolinol, was found to determine the level of the neurotoxin in the brain. The activity of neutral(R)salsolinol N-methyltransferase was examined using lymphocytes prepared from PD patients, normal controls and diseased controls as enzyme source. A significant increase in the activity was confirmed in lymphocytes from PD cases compared to normal- and diseased-control. Studies to clarify the environmental and genetic factors determining the activity of the enzyme are now under the way. The cytotoxicity of N-methyl(R)salsolinol was examined using a cultured cell model. N-Methyl(R)salsolinol was found to induce apoptotic cell death in a dose-dependent way. The mechanism of apoptosis was clarified to be mediated by collapse in mitochondrial membrane potential, activation of caspase 3 and fragmentation of nuclear DNA. In addition, propargylamines protected the cells from apoptosis. It was suggested that N-methyl(R)salsolinol and propargylamines have specific binding sites in mitochondria which regulate the death signal transduction. Propargylamines might be applicable as neuroprotective drugs, which can be orally administrated to PD patients.