Specificity of Pyridinium Inhibitors of the Ubiquinone Reduction Sites in Mitochondrial Complex I

Abstract

I) 1 is a large enzyme that catalyzes the oxidation of NADH by ubiquinone coupled to proton translocation across the inner membrane (1, 2). There are a variety of inhibitors of mitochondrial complex I and with the exception of a few inhibitors which inhibit electron input into the enzyme (3, 4), all inhibitors act at or close to the ubiquinone reduction site (5). Among the inhibitors, positively charged neurotoxic N-methyl-4-phenylpyridinium (MPP ϩ ) and its alkyl analogues exhibit unique inhibitory behavior with bovine heart mitochondrial complex I (6). A series of studies of the inhibition mechanism of MPP ϩ analogues by Singer and colleagues (6 -10) have suggested that MPP ϩ analogues are bound at two sites in the enzyme, one accessible to relatively hydrophilic inhibitors (termed the "hydrophilic site") and one shielded by a hydrophobic barrier on the enzyme (the "hydrophobic site"), and that occupation of both sites is required for complete inhibition. This concept may be helpful in elucidating the terminal electron transfer step in complex I and seems to be consistent with the existence of two EPR-detectable species of complex I-associated ubisemiquinones (11,12). Some experimental results with ordinary complex I inhibitors (13-16) can be explained by assuming the existence of more than one inhibitor (or ubiquinone) binding site.In the previous study (17), we synthesized a series of MPP ϩ analogues which are much more potent than the original MPP ϩ and demonstrated that the presence of hydrophobic counteranion tetraphenylboron (TPB Ϫ ) potentiates the inhibition by MPP ϩ analogues differently depending upon the molar ratio of TPB Ϫ to the inhibitors. In the presence of a catalytic amount of TPB Ϫ , the inhibitory potency of MPP ϩ analogues was markedly enhanced, and the extent of inhibition was almost complete. The presence of an excess amount of TPB Ϫ partially reactivated the enzyme activity, and the inhibition was partly saturated (ϳ50%). This complicated inhibitory behavior could be explained by the dual binding sites model mentioned above (6), which supposes quite different hydrophobic natures of the two sites and/or their environments.If there are indeed two distinct binding sites of MPP ϩ analogues in bovine complex I, there should be specific inhibitors which act selectively at one of the two proposed binding sites since it is unlikely that the structural properties of the two sites are completely identical. We have synthesized such a selective inhibitor, MP-6 (N-methyl-4-[2-(p-tert-butylbenzyl)-propyl]pyridinium, Fig. 1) (17). In the absence of TPB Ϫ , this inhibitor showed approximately 50% inhibition at 5 M in NADH-Q 1 oxidoreductase assay, but the inhibition reached a plateau at this level over a wide range of concentrations. Weak inhibition was again observed above ϳ80 M, and maximum inhibition (Ͼ90%) was obtained only when the concentration of the inhibitor was increased to ϳ250 M. Such a marked biphasic nature of the does-response curve has not been reported previously for usual complex I i...

Design syntheses and mitochondrial complex I inhibitory activity of novel acetogenin mimics

“…Interestingly, this appeared to be true also for the case of acetogenins, the most potent inhibitors of the enzyme. The loose recognition by the enzyme of the inhibitor structure may reflect the large cavity‐like structure of the inhibitor (or ubiquinone) binding domain in complex I [8,9,27,34,35]. Nevertheless, the present study provided useful guiding principles for simplification of acetogenin structures and will save a great deal of labour involving stereoselective syntheses of the stereogenic moieties.…”

“…It therefore remains to be elucidated which ring moiety (the γ‐lactone or the bis‐THF ring) shares the same binding domain with the ordinary inhibitors. Taking into account the proposal of a fairly large binding domain of inhibitor (or ubiquinone) in complex I [8,9,27,34,35], the possibility that both ring moieties occupy different subsites in the same binding domain, in analogy with different types of Q o centre inhibitors of cytochrome bc 1 complex [36], by the support of some folded‐conformation of the alkyl spacer cannot be excluded.…”

“…On the basis of a number of structure–activity relationship studies of various complex I inhibitors (e.g. [34,35,37,38]), one of the authors (H.M.) noted in a recent review that in contrast with complex III inhibitors, essential structural factors of complex I inhibitors that drastically affect the inhibitory potency are not necessarily obvious [10]. Interestingly, this appeared to be true also for the case of acetogenins, the most potent inhibitors of the enzyme.…”

Fe‐Catalyzed Intramolecular Cross‐Dehydrogenative Arylation (CDA), Efficient Synthesis of 1‐Arylnaphthalenes and 4‐Arylcoumarins

“…The yield of 1,4-di-o-tolylbutane 1g was slightly lower (45 % isolated yield) than its para analog 1b, while they all met the same over-oxidize issue. Then, two asymmetric 1,4-diphenylbutane substrates 1h and 1i were synthesized [76,77] and introduced to this system. For the reaction of 1-(tert-butyl)-4-(2-methyl-4-phenylbutyl)benzene (1h), fascinating, good regio selectively was observed.…”

Neuroprotection or Neurotoxicity of Illicit Drugs on Parkinson’s Disease

“…The relationship between these contaminants and PD has already been described. MPPP, a heroin analogue, is quickly converted in its metabolite MPP + , which promotes a syndrome indistinguishable from Parkinsonism, after cell uptake by the dopamine transporter of dopaminergic neurons inhibiting the activity of the mitochondrial nicotinamide adenine dinucleotide hydride (NADH)-Q dehydrogenase complex (EC 1.6.5.3) [ 284 , 285 , 286 , 287 ]. Moreover, MPTP also destroys dopamine-making cells in substantia nigra [ 283 ].…”