Pramod K. Arora

Inhibition of mitochondrial respiration by analogs of 4-phenylpyridine and 1-methyl-4-phenylpyridinium cation (MPP+), the neurotoxic metabolite of MPTP

In order to clarify the structural requirements associated with the inhibition of mitochondrial respiration by MPP+, the neurotoxic metabolites of the Parkinsonian agent MPTP, ten sets of pyridine/N-methylpyridinium pairs and a few miscellaneous compounds were evaluated on rat liver intact mitochondria (Mw) and on submitochondrial particles (SMP). The pyridinium partners were much more potent inhibitors on Mw than on SMP, indicating that they are concentrated inside mitochondria by the energy-dependent process previously reported for MPP+, probably as a consequence of non-specific passive transport across the mitochondrial inner membrane in response to the transmembrane potential. In the SMP assay, the neutral pyridines were stronger inhibitors than were the pyridinium cations, and the inhibitory potency varied little with structural changes. The N-methylated forms of beta-carbolines may act as endogenous MPP+-like agents.

Model study on the bioreduction of paraquat, MPP+, and analogs. Evidence against a “redox cycling” mechanism in MPTP neurotoxicity

The ability of paraquat, MPP+, and analogs to be reduced by chemical reductants and by NADPH, as catalyzed by liver microsomes or purified NADPH cytochrome P-450 reductase, is reported. The analogs span a range of electrochemical potential, including values in-between that of paraquat and MPP+. Analogs with an Eo below -.55 V (vs. NHE) are not reduced by either the NADPH-microsomes or NADPH-reductase systems. The inability of MPP+ to be bio-reduced or to stimulate the production of superoxide during aerobic reduction is evidence against a redox-cycling (oxidant stress) role of MPP+ in MPTP neurotoxicity.

Dopaminergic Neurotoxicity In Vivo and Inhibition of Mitochondrial Respiration In Vitro by Possible Endogenous Pyridinium-Like Substances

Elucidation of the mechanism(s) by which 1‐methyI‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its active metabolite l‐methyl‐4‐phenylpyridinium (MPP+) cause parkinsonism in humans and other primates has prompted consideration of possible endogenous MPTP/MPP+‐like ncurotoxins in the etiology of idiopathic Parkinsons disease. Here we examined inhibition of mitochondrial respiration in vitro and neurotoxicity in rats in vivo produced by β‐carbolinium compounds that are presumed to form following Pictet‐Spengler cyclization of serotonin. We also evaluated N‐methyiisoquinolinium, a putative endogenous neurotoxin, in the same manner. The latter compound exhibited MPP+ like mitochondrial respiratory inhibition, whereas the β‐carbotinitim compounds, although more potent inhibitors of electron transport, exhibited weak accumulation‐ dependent enhancement of inhibition in intact mitochondria. It is interesting that the β‐carbolinium compounds inhibited succinate‐as well as glutamate‐supported respiration, and are best described as inhibitor‐uncouplers. The results of partitioning experiments suggest that both the low accumulation potential and the inhibition of succinate respiration may be a consequence of the β‐carboliniums being in equilibrium with neutral “anhydro” bases. Relative to MPP+, all compounds tested had weak dopaminergic uptake activity in vitro and weak dopaminergic toxicity in vivo, consistent with other findings of relatively low neurotoxic potential for presumed endogenous pyridiniums.

Comparative toxicity of MPTP, MPP+ and 3,3-dimethyl-MPDP+ to dopaminergic neurons of the rat substantia nigra

The depletion of dopamine and its metabolites in the striatum of rats was assessed 3 weeks after the unilateral intranigral infusion of different doses of MPTP, its ultimate metabolite MPP+, and 3,3-DM-MPDP+, a non-oxidizable analog of the initial MPTP metabolite 2,3-MPDP+. MPP+ was found to be 2-3 orders of magnitude more toxic than the other two agents, consistent with the view that it is primarily responsible for MPTP neurotoxicity.

Mechanism of neurotoxic action of β,β′-iminodipropionitrile (IDPN): N-hydroxylation enhances neurotoxic potency

The molecular mechanism(s) whereby β,β′-iminodipropionitrile (IDPN) induces an excitatory behavioral syndrome and a distinct alteration of the axonal cytoskeleton in experimental animals is not known. We demonstrate here that upon intraperitoneal administration to rats, the N-hydroxy analog of IDPN (HOIDPN) induces a parallel spectrum of both neurotoxic effects of IDPN and is approximately 8 times more potent than IDPN in this regard. This is consistent with the involvement of a flavin monooxygenase-mediated N-oxygenation pathway in the toxic activation of IDPN.

Structure-neurotoxicity trends of analogues of 1-methyl-4-phenylpyridinium (MPP+), the cytotoxic metabolite of the dopaminergic neurotoxin MPTP

The dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) derives from its metabolism to 1-methyl-4-phenyl-pyridinium cation (MPP+), which is then selectively accumulated in dopaminergic neurons. In an effort to assess the structural requirements governing MPP+ cytotoxicity, we evaluated dopaminergic toxicity of MPP+ analogues 3 weeks after their microinfusion into rat substantia nigra. We also evaluated the substrate suitability of MPP+ analogues for high-affinity dopamine uptake in striatal synaptosomes by measuring their ability to induce specific dopamine release. The intranigral neurotoxicity of MPP+ analogues in vivo correlates mainly with their in vitro inhibitory activity on mitochondrial respiration, consistent with a compromise in cellular energy production as the principal mechanism of MPTP-induced cell death. This study extends the structure-neurotoxicity data base beyond that obtainable using MPTP analogues, since many of these are not metabolized to pyridinium compounds. Such information is crucial to assess which possible endogenous or exogenous compounds may exert MPTP/MPP(+)-like toxicity.

Chemical oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its in vivo metabolism in rat brain and liver.

We investigated in vivo the metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the brain and liver of rats 45 min after the systemic administration of 50 mg/kg of the neurotoxin. The metabolites present in brain and liver extracts were identified through multiple analytical methods by comparison to authentic compounds obtained from a number of chemical oxidations of MPTP. Our results indicate the presence of approximately 15% unreacted MPTP and relatively large amounts of both 1-methyl-4-phenylpyridinium (MPP+) and a mixture of three nonpolar lactams: 1-methyl-4-phenyl-5,6-dihydro-2(1H)-pyridinone, 1-methyl-4-phenyl-2(1H)-pyridinone, and a previously unreported metabolite 1-methyl-4-phenyl-2-piperidinone. Whereas MPP+ was more prevalent in the brain than in the liver, the lactam metabolites were more predominant in the liver. The amounts of the N-oxide and N-demethylated metabolites of MPTP were minimal.

Inhibition of mitochondrial respiration by analogues of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium: Structural requirements for accumulation-dependent enhanced inhibitory potency on intact mitochondria☆

Analogues of 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, were evaluated for inhibition of respiration in intact mitochondria (Mw) and in electron transport particles (ETP). MPP+ exhibits relatively weak inhibitory activity in ETP, but potent inhibition in Mw occurs on account of its energy-dependent accumulation inside mitochondria. The permeant anion tetraphenylborate potentiates the inhibition in both Mw and ETP. Replacement of the 4-phenyl ring of MPP+ by a variety of aromatic and nonaromatic rings, and of the N-methylpyridinium group by other cationic aromatic heterocycles, preserves the inhibitory patterns seen for MPP+. The general observation of enhanced inhibitory potency in Mw for all these permanently charged cations is consistent with our contention that energy-dependent accumulation inside mitochondria represents a passive Nernstian concentration in response to the transmembrane electrochemical gradient. Nonetheless, the magnitude of the inhibitory potentiation seen in Mw relative to ETP varies widely with structure. In particular, less lipophilic analogues, especially those bearing a localized, rather than resonance-stabilized, permanent positive charge, exhibit similar inhibitory activity to MPP+ in ETP, but the inhibition in Mw is not comparably enhanced. For these same analogues, the inhibitory activity in ETP is only weakly potentiated by tetraphenylborate. Since succinate was found to completely reverse the respiratory inhibition in Mw induced by all types of MPP+ analogues investigated, a common site 1 inhibition appears to be involved; thus the different inhibitory patterns observed must be due to structural factors governing membrane transport and distribution properties.

Potent Neurotoxic Fluorinated 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Analogs as Potential Probes in Models of Parkinson Disease

Abstract: We synthesized a number of fluorinated analogs of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), and tested their suitability as substrates for monoamine oxidase B in vitro and their dopaminergic neurotoxicity in vivo. Two of the compounds tested, 2′‐F‐MPTP and 2′‐CF3‐MPTP, were better enzyme substrates and possessed more potent neurotoxicity for nigrostriatal dopamine neurons than MPTP, especially 2′‐F‐MPTP. The results of the in vivo neurotoxicity experiments correlated well with the suitability of the compounds as substrates for monoamine oxidase. These findings could serve as a basis for the use of 18F‐labeled analogs of MPTP for positron emission tomography studies of nonhuman primates for better understanding of the factors underlying MPTP toxicity. Furthermore, the discovery of two MPTP analogs with enhanced selective neurotoxicity to dopaminergic neurons may be an important clue in the continuing efforts to define the chemical structure‐activity factors governing MPTP neurotoxic activation mechanisms.

Copper-ammonia mediated oxidation of carbonyl compounds

Copper(II) in aqueous NH4OH, in most cases in conjunction with O2 or K2S2O8 as auxiliary oxidant, induces oxidative cleavage of ketones to nitriles when there is a good carbon electrofuge; for unactivated ketones, the main reaction observed is oxidative coupling of ketimines to give azines. If R′ = poor electrofuge, oxidative coupling to RR′ C=N-N=CRR′ can occur.