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

Hideto MiyoshiDagger , Jun Iwata, Kimitoshi Sakamoto, Hiroshi Furukawa, Motoyuki Takada, Hajime Iwamura, Takashi Watanabe§, and Yoshio Kodama§

From the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan and the § Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Kohoku-ku, Yokohama 222-8567, Japan

    ABSTRACT
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Results & Discussion
References

Dual binding sites for pyridinium-type inhibitors in bovine heart mitochondrial complex I have been proposed (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167-3174). The marked biphasic nature of the dose-response curve for inhibition of the enzyme by MP-6(N-methyl-4-[2-(p-tert-butylbenzyl)propyl]pyridinium) makes this compound the first selective inhibitor of the two sites (Miyoshi, H., Inoue, M., Okamoto, S., Ohshima, M., Sakamoto, K., and Iwamura, H. (1997) J. Biol. Chem. 272, 16176-16183). Modifications of the structure of MP-6 show that a tert-butyl group on the benzene ring, a methyl group attached to the pyridine nitrogen atom, para-substitution pattern in the pyridine ring, and the presence of a branched structure in the spacer moiety are important for the selective inhibition. On the basis of the structural specificity, we synthesized a selective inhibitor, MP-24 (N-methyl-4-[2-methyl-2-(p-tert-butylbenzyl)propyl]pyridinium), which elicits greater selectivity. Characterization of the inhibitory behavior of MP-24 provided further strong evidence for the dual binding sites model.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Mitochondrial NADH-ubiquinone oxidoreductase (complex 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+) 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+ analogues by Singer and colleagues (6-10) have suggested that MPP+ 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+ analogues which are much more potent than the original MPP+ and demonstrated that the presence of hydrophobic counter-anion tetraphenylboron (TPB-) potentiates the inhibition by MPP+ analogues differently depending upon the molar ratio of TPB- to the inhibitors. In the presence of a catalytic amount of TPB-, the inhibitory potency of MPP+ analogues was markedly enhanced, and the extent of inhibition was almost complete. The presence of an excess amount of TPB- partially reactivated the enzyme activity, and the inhibition was partly saturated (~50%). 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+ 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-, this inhibitor showed approximately 50% inhibition at 5 µM in NADH-Q1 oxidoreductase assay, but the inhibition reached a plateau at this level over a wide range of concentrations. Weak inhibition was again observed above ~80 µM, and maximum inhibition (>90%) was obtained only when the concentration of the inhibitor was increased to ~250 µM. Such a marked biphasic nature of the does-response curve has not been reported previously for usual complex I inhibitors. On the other hand, almost complete inhibition was readily obtained at low concentrations of MP-6 (<10 µM) in the presence of 2 µM TPB-. The site that is readily blocked by low concentrations of MP-6 without TPB- would be the hydrophilic binding site. Thus, MP-6 is a fairly selective inhibitor of one of the two proposed binding sites and could be a useful probe to examine the mechanism of the terminal electron transfer step of complex I. This inhibitor is, however, not completely selective in the strict sense because it elicits complete inhibition at high concentrations without TPB-, as mentioned above. Therefore, highly selective pyridinium-type inhibitors superior to MP-6 are required.


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Fig. 1.   Structures of pyridinium- and quinolinium-type inhibitors examined in this study. The compound numbers are given referring to our previous work (17).

Among the 19 cationic inhibitors synthesized previously (17), only MP-6 exhibited such a unique inhibitory behavior, suggesting that some specific structural feature(s) of this compound would be responsible for its behavior. Elucidation of such structural features is essential to develop further selective inhibitors. In the present study, we systematically modified the structure of the lead compound MP-6 (Fig. 1) and investigated the effects of structural modifications on its inhibitory behavior. On the basis of the structural information, we synthesized a selective inhibitor superior to MP-6. Characterization of the inhibitory behavior of the new inhibitor provides further evidence for the dual binding sites model.

    EXPERIMENTAL PROCEDURES
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References

Materials-- Q1 was a generous gift from Eisai Co. (Tokyo, Japan). Piericidin A was generously provided by Dr. Shigeo Yoshida (RIKEN, Japan). Other chemicals were commercial products of analytical grade.

Synthesis-- All synthetic compounds were characterized by 1H NMR spectroscopy (Bruker ARX-300) and elemental analyses for carbon, hydrogen, and nitrogen, within an error of ± 0.3%. N-Methylation (or N-ethylation) of the compounds was carried out by the previously reported method (17).

Synthesis of MP-6 and Its Derivatives-- The previous synthetic method for MP-6 (17) was not suitable for preparation of a variety of derivatives because of low reaction yield and limits of structural variations of starting materials. We therefore synthesized MP-6 by a new method as follows (Method 1 in Fig. 2).


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Fig. 2.   Synthetic procedures. a, n-propionylchloride, AlCl3 in CH3NO2 at room temperature; b, 4-pyridinecarboxaldehyde, lithium diisopropylamide in THF at -78 °C; c, methylsulfonyl chloride/Et3N in THF at 0 °C; d, reflux in pyridine; e, H2, Pd/C in EtOH at room temperature; f, (NH2)2, KOH in ethylene glycol at 150 °C; g, methyliodide, NaH in THF at room temperature; h, p-tert-butylbenzylbromide, lithium diisopropylamide in THF at room temperature.

To a mixture of tert-butylbenzene (16 g, 0.12 mol) and n-propionyl chloride (9.3 g, 0.1 mol), was added aluminum chloride (14.7 g, 0.11 mol) slowly at 0 °C. After stirring at room temperature for 3 h, the mixture was poured into ice water and extracted with ether, washed with 1 N HCl, 1 N NaOH, and brine, dried over MgSO4, and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 1:20) to give 4-propionyl-tert-butylbenzene in a 47% yield.

To a solution of lithium diisopropylamide in THF, was added 4-propionyl-tert-butylbenzene (7.7 g, 40.5 mmol) slowly at -78 °C and the reaction mixture was stirred for 1 h. After adding 4-pyridinecarboxaldehyde (4.4 g, 41 mmol) dropwise, the mixture was stirred for 1 h. The reaction was quenched with saturated aqueous NH4Cl, ether was added, and the organic phase was washed with brine and dried over MgSO4. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1) to give 4-[1-hydroxy-2-(p-tert-butylbenzoyl)propyl]pyridine in a 90% yield.

To a solution of 4-[1-hydroxy-2-(p-tert-butylbenzoyl)propyl]pyridine (3.3 g, 11.1 mmol) and triethylamine (2.5 g, 24.7 mmol) in 30 ml of THF, methanesulfonyl chloride (1.4 g, 12.2 mmol) was added slowly at 0 °C. After stirring at 0 °C for 30 min, the reaction mixture was extracted with ether, washed with saturated Na2CO3 and brine, then dried over MgSO4, and concentrated. The residue 4-[1-methanesulfonyloxy-2-(p-tert-butylbenzoyl)propyl]pyridine was used directly in the next step.

4-[1-Methanesulfonyloxy-2-(p-tert-butylbenzoyl)propyl]pyridine was dissolved in 20 ml of pyridine and the reaction mixture was refluxed overnight. The mixture was evaporated and then the residue was extracted with ether, washed with brine, dried over MgSO4, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 3:7) to give 4-[2-(p-tert-butylbenzoyl)propenyl]pyridine in a 93% yield.

To a solution of 4-[2-(p-tert-butylbenzoyl)propenyl]pyridine (1.0 g, 3.6 mmol) in 10 ml of ethanol, Pd/C (0.1 g, 10% w/w) was added and the reaction mixture was stirred under H2, until TLC analysis failed to reveal the presence of the precursor. Pd/C was removed by celite filtration, and the solvent was evaporated. Purification by silica gel column chromatography (ethyl acetate/hexane, 3:7) gave 4-[2-(p-tert-butylbenzoyl)propyl]pyridine in a 89% yield.

To a solution of 4-[2-(p-tert-butylbenzoyl)propyl]pyridine (0.8 g, 2.8 mmol) in 8 ml of ethylene glycol, hydrazine monohydrate (0.42 g, 8.4 mmol) and KOH were added (0.55 g, 8.4 mmol). The reaction mixture was stirred at 150 °C for 5 h. The mixture was extracted with ether, washed with brine, dried over MgSO4, and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 1:4) to give 4-[2-(p-tert-butylbenzyl)propyl]pyridine in a 66% yield. MP-6: 1H NMR (CDCl3, 300 MHz) delta  0.89 (d, J = 6.6 Hz, 3H), 1.31 (s, 9H), 2.20 (m, 1H), 2.52-2.67 (m, 3H), 2.95 (m, 1H), 4.41 (s, 3H), 7.07 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 6.6 Hz, 2H), 8.70 (d, J = 6.6 Hz, 2H).

MP-11 and MP-12 were prepared by the same method as used for MP-6, except that p-n- or p-sec-butylbenzene was used, respectively, in place of p-tert-butylbenzene in step a. MP-11: 1H NMR (CDCl3, 300 MHz) delta  0.86 (m, 3H), 0.92 (m, 3H), 1.35 (m, 2H), 2.57 (m, 4H), 2.18 (m, 1H), 2.59 (m, 4H), 4.42 (s, 3H), 7.01 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 6.6 Hz, 2H), 8.69 (d, J = 6.6 Hz, 2H). MP-12: 1H NMR (CDCl3, 300 MHz) delta  0.81 (t, J = 7.3 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H), 1.22 (d, J = 6.9 Hz, 3H), 1.57 (d, J = 7.3 Hz, 2H), 2.17 (m, 1H), 2.58 (m, 1H), 2.92 (m, 1H), 4.41 (s, 3H), 7.04 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 6.6 Hz, 2H), 8.71 (d, J = 6.6 Hz, 2H).

MP-14, MP-15, and MQ-22 were prepared by the same method for MP-6, except that 2-pyridinecarboxaldehyde, 3-pyridinecarboxaldehyde, or 4-quinolinecarboxaldehyde was used, respectively, in place of 4-pyridinecarboxaldehyde in the step b. MP-14: 1H NMR (CDCl3, 300 MHz) delta  1.06 (d, J = 6.6 Hz, 3H), 1.30 (s, 9H), 2.24 (m, 1H), 2.57 (m, 1H), 2.80 (m, 2H), 3.13 (m, 1H), 4.14 (s, 3H), 7.09 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 6.4 Hz, 1H), 8.24 (t, J = 7.9 Hz, 1H), 8.90 (d, J = 6.2 Hz, 1H). MP-15: 1H NMR (CDCl3, 300 MHz) delta  0.90 (d, J = 6.6 Hz, 3H), 1.30 (s, 9H), 2.15 (m, 1H), 2.61 (m, 3H), 2.91 (m, 1H), 4.44 (s, 3H), 7.06 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 7.84 (t, J = 7.6 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.61 (s, 1H), 8.71 (d, J = 5.6 Hz, 1H). MQ-22: 1H NMR (CDCl3, 300 MHz) delta  0.99 (d, J = 6.6 Hz, 3H), 1.34 (s, 9H), 2.20 (m, 1H), 2.63 (m, 1H), 2.86 (m, 1H), 3.50 (m, 1H), 4.62 (s, 3H), 7.14 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 7.6 Hz, 2H), 7.78 (m, 2H), 7.86 (d, J = 8.3 Hz, 1H), 8.14 (t, J = 6.5 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 9.16 (d, J = 6.0 Hz, 1H).

MP-17 was prepared by the same method as used for MP-6, except that 4-acetyl-tert-butylbenzene was used in place of 4-propionyl-tert-butylbenzene in step b. MP-17: 1H NMR (CDCl3, 300 MHz) delta  1.31 (s, 9H), 2.00-2.10 (m, 2H), 2.69 (t, J = 7.3 Hz, 2H), 2.89 (t, J = 7.8 Hz, 2H), 4.44 (s, 3H), 7.09 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 6.5 Hz, 2H), 8.70 (d, J = 6.4 Hz, 2H).

MP-19 and MP-20 were obtained as reaction intermediates of MP-6 and MP-22, respectively. MP-19: 1H NMR (CDCl3, 300 MHz) delta  1.32 (s, 9H), 1.34 (d, J = 7.2 Hz, 3H), 3.00 (m, 1H), 3.46 (m, 1H), 3.96 (m, 1H), 4.37 (s, 3H), 7.48 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 6.5 Hz, 2H), 8.66 (d, J = 6.6 Hz, 2H). MP-20: 1H NMR (CDCl3, 300 MHz) delta  1.34 (s, 9H), 2.02 (d, J = 7.1 Hz, 3H), 4.04 (s, 2H), 6.85 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 6.8 Hz, 2H), 8.69 (d, J = 6.8 Hz, 2H).

MP-23 was prepared by the same method as used for MP-6, except that n-butyrylbromide was used in place of n-propionylchloride in step a. MP-23: 1H NMR (CDCl3, 300 MHz) delta  0.96 (t, J = 7.3 Hz, 3H), 1.30 (s, 9H), 1.33 (m, 2H), 2.08 (m, 1H), 2.39 (m, 2H), 2.77 (m, 2H), 4.39 (s, 3H), 7.03 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 6.6 Hz, 2H), 8.66 (d, J = 6.6 Hz, 2H).

Synthesis of MP-24 and Its Derivatives-- MP-24 was synthesized by Method 2 as shown in Fig. 2. To a suspension of NaH (2, 2 g, 91.0 mmol) in 50 ml of THF, 4-acetylpyridine (5.0 g, 41.3 mmol) was added slowly at 0 °C under N2 and the mixture was stirred for 30 min, after which methyliodide (12.8 g, 90.0 mmol) was added and stirred at room temperature overnight. The reaction mixture was extracted with ether and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 3:7) to give 4-(2-methylpropionyl)pyridine in a 56% yield.

To a solution of diisopropylamine (0.8 g, 7.4 mmol) in 20 ml of THF, 4.6 ml of 1.6 M n-butyllithium (7.4 mmol) was added slowly at 0 °C under N2 and stirred for 30 min, after which hexamethylphosphoric triamide (1.3 g, 7.4 mmol) was added and stirred for 30 min. This reaction mixture was cooled to -78 °C, after which 4-(2-methylpropionyl)pyridine (1.0 g, 6.7 mmol) was added slowly and stirred for 1 h. After adding p-tert-butylbenzylbromide (1.7 g, 7.4 mmol) dropwise, the mixture was slowly warmed to room temperature. Extraction with ether, washing with brine, and drying over MgSO4 gave the crude product, which was purified by silica gel column chromatography (ethyl acetate/hexane, 3:7) to give 4-[2-methyl-2-(p-tert-butylbenzyl)propionyl]pyridine in a 34% yield.

To a solution of 4-[2-methyl-2-(p-tert-butylbenzyl)propionyl]pyridine (0.7 g, 2.3 mmol) in 10 ml of ethylene glycol, hydrazine monohydrate (0.4 g, 7.0 mmol) and KOH (0.4 g, 7.0 mmol) were added and stirred at 150 °C for 5 h. The mixture was extracted with ether and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 3:7) to give 4-[2-methyl-2-(p-tert-butylbenzyl)propyl]pyridine (MP-24) in a 56% yield. MP-24: 1H NMR (CDCl3, 300 MHz) delta  0.89 (s, 6H), 1.32 (s, 9H), 2.61 (s, 2H), 2.78 (s, 2H), 4.46 (s, 3H), 7.05 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 7.67 (d, J = 6.6 Hz, 2H), 8.72 (d, J = 6.6 Hz, 2H).

MP-25 was synthesized by the same method as used for MP-24, except that 2-acetylpyridine was used in place of 4-acetylpyridine in step g. MP-25: 1H NMR (CDCl3, 300 MHz) delta  0.99 (s, 6H), 1.32 (s, 9H), 2.71 (s, 2H), 3.12 (s, 2H), 4.33 (s, 3H), 7.09 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 6.5 Hz, 2H), 7.69 (d, J = 6.7 Hz, 1H), 7.87 (t, J = 6.2 Hz, 1H), 8.28 (t, J = 7.0 Hz, 1H), 9.02 (d, J = 5.7 Hz, 1H).

MP-26 and MP-27 were synthesized by the same method as used for MP-24, except that p-n-butylbenzyliodide or p-tert-amylbenzyliodide were used, respectively, in place of p-tert-butylbenzylbromide in step h. MP-26: 1H NMR (CDCl3, 300 MHz) delta  0.89 (s, 6H), 0.93 (t, J = 7.3 Hz, 3H), 1.36 (m, 2H), 1.59 (m, 2H), 2.60 (s, 2H), 2.60 (t, J = 5.7 Hz, 2H), 2.77 (s, 2H), 4.46 (s, 3H), 7.02 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 6.6 Hz, 2H), 8.71 (d, J = 6.5 Hz, 2H). MP-27: 1H NMR (CDCl3, 300 MHz) delta  0.68 (t, J = 7.4 Hz, 3H), 0.89 (s, 6H), 1.28 (s, 6H), 1.63 (q, J = 7.5 Hz, 2H), 2.60 (s, 2H), 2.78 (s, 2H), 4.45 (s, 3H), 7.05 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 6.7 Hz, 2H), 8.72 (d, J = 6.5 Hz, 2H).

Syntheses of Other Compounds-- MP-21 and MQ-20 were prepared by reacting 4-chloropyridine or 4-chloroquinoline with 2-methyl-3-(p-tert-butylphenyl)propanol, respectively, in the presence of sodium and a catalytic amount of KI in 50% dimethyl sulfoxide/THF at 70 °C. 2-Methyl-3-(p-tert-butylphenyl)propanol was prepared from the corresponding propionaldehyde by the previously reported method (17). MP-21: 1H NMR (CDCl3, 300 MHz) delta  1.08 (d, J = 6.7 Hz, 3H), 1.30 (s, 9H), 2.65 (m, 2H), 4.08 (m, 2H), 4.30 (s, 3H), 7.07 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 7.5 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 8.61 (d, J = 7.1 Hz, 2H). MQ-20: 1H NMR (CDCl3, 300 MHz) delta  1.20 (d, J = 6.8 Hz, 3H), 1.29 (s, 9H), 2.52 (m, 1H), 2.75 (m, 2H), 4.33 (m, 2H), 4.53 (s, 3H), 7.11 (d, J = 8.3 Hz, 2H), 7.33 (m, 3H), 7.87 (m, 1H), 8.13 (m, 2H), 8.41 (d, J = 8.3 Hz, 1H), 8.44 (d, J = 7.1 Hz, 1H).

MP-22 and MQ-21 were prepared reacting 4-methylpyridine or 4-methylquinoline and 1-iodo-2-methyl-3-(p-tert-butylphenyl)propane, respectively, according to the previously reported method for MQ-14 (17). 1-Iodo-2-methyl-3-(p-tert-butylphenyl)propane was obtained by reacting 2-methyl-3-(p-tert-butylphenyl)propanol with triphenylphosphonate and methyliodide in THF at 70 °C (18). MP-18 was synthesized under the same reaction conditions, but using 4-methylpyridine and p-tert-butylbenzylbromide. MP-22: 1H NMR (CDCl3, 300 MHz) delta  0.98 (d, J = 6.5 Hz, 3H), 1.31 (s, 9H), 1.52 (m, 1H), 1.72-1.84 (m, 2H), 2.78-3.00 (m, 2H), 4.45 (s, 3H), 7.05 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 6.6 Hz, 2H), 8.70 (d, J = 6.6 Hz, 2H). MQ-21: 1H NMR (CDCl3, 300 MHz) delta  1.07 (d, J = 6.6 Hz, 3H), 1.31 (s, 9H), 1.64 (m, 1H), 1.79-2.00 (m, 2H), 2.50-2.69 (m, 2H), 3.11-3.39 (m, 2H), 4.64 (s, 3H), 7.09 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 6.1 Hz, 1H), 7.89 (t, J = 8.1 Hz, 1H), 8.13-8.22 (m, 2H), 8.32 (d, J = 8.8 Hz, 1H), 9.24 (d, J = 6.1 Hz, 1H). MP-18: 1H NMR (CDCl3, 300 MHz) delta  1.30 (s, 9H), 3.00 (t, J = 7.6 Hz, 2H), 3.20 (t, J = 7.6 Hz, 2H), 4.45 (s, 3H), 7.06 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 6.7 Hz, 2H), 8.70 (d, J = 6.6 Hz, 2H).

MP-16 was prepared by reduction of 4-[(2-carboxy-3-p-tert-butylphenyl)propyl]pyridine with LiAlH4 in THF. 4-[(2-Carboxy-3-p-tert-butylphenyl)propyl]pyridine was prepared by decarboxylation of 4-[(2,2-diethoxycarbonyl-3-p-tert-butylphenyl)propyl]pyridine, which was obtained by reacting 4-[(2,2-diethoxycarbonyl)ethyl]pyridine and p-tert-butylbenzylbromide in the presence of NaH in THF at 0 °C. 4-[(2,2-Diethoxycarbonyl)ethyl]pyridine was prepared by reducing 4-[(2,2-diethoxycarbonyl)vinyl]pyridine, which was synthesized by reacting 4-pyridinecarboxaldehyde and diethylmalonate in the presence of a catalytic amount of piperidine at 100 °C, with 10% Pd/C in ethanol under hydrogen gas at room temperature. MP-16: H NMR (CDCl3, 300 MHz) delta  1.28 (s, 9H), 2.30 (m, 1H), 2.50 (m, 1H), 2.82 (m, 2H), 3.45 (m, 1H), 4.28 (s, 3H), 4.35 (d, J = 2.1 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H0, 7.69 (d, J = 6.6 Hz, 2H), 8.48 (d, J = 6.6 Hz, 2H).

MP-28 was synthesized by the same method as used for 4-[2,2-diethoxycarbonyl-3-(p-tert-butylphenyl)propyl]pyridine, which was an intermediate of MP-16, except that acetylacetone was used in place of diethylmalonate. MP-28: 1H NMR (CDCl3, 300 MHz) delta  1.33 (s, 9H), 2.35 (s, 6H), 3.48 (s, 2H), 3.53 (s, 2H), 4.33 (s, 3H), 7.09 (m, 2H), 7.37 (m, 2H), 7.76 (m, 2H), 8.46 (m, 2H).

Methods-- Bovine heart submitochondrial particles were prepared by the method of Matsuno-Yagi and Hatefi (19) and stored in a buffer containing 0.25 M sucrose and 10 mM Tris/HCl (pH 7.4) at -78 °C. NADH-Q1 oxidoreductase activity was measured as the rate of NADH oxidation with a Shimadzu UV-3000 at 340 nm (epsilon  = 6.2 mM-1 cm-1). The reaction medium contained 50 mM potassium phosphate (pH 7.4), 0.25 M sucrose, 1 mM MgCl2, 2 mM KCN, 0.4 µM antimycin A, and the final mitochondrial protein concentration was 30 µg/ml. The reaction was started by adding 50 µM NADH after submitochondrial particles were incubated with inhibitor for 4 min at 30 °C. Q1 (50 µM) was used as an electron acceptor since this substrate is the best electron acceptor, yielding high reaction rate with linear kinetics (20). Unless otherwise noted, the concentration of TPB- was set at 2 µM throughout the present study since the inhibition by TPB- itself was not negligible above this concentration. The experimental conditions for investigation of the effects of incubation temperature or incubation time on the inhibition by MP-24 are described in the figure legends.

Ferricyanide and dichloroindophenol reduction by complex I was assayed at 420-500 and 600 nm, respectively, under the same experimental conditions. When ferricyanide (1 mM) or dichloroindophenol (50 µM) was used as an electron acceptor, the concentration of NADH was set at 150 or 50 µM, respectively.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Effects of Structural Modification on Inhibitory Behavior of MP-6-- The I75/I25 ratio was used as an index of the biphasic nature of the titration curve (Table I), wherein the I75 and I25 were the molar concentrations to give 75 and 25% inhibition of the control NADH-Q1 oxidoreductase activity without TPB-, respectively. The I50 values, i.e. the molar concentrations which gave 50% inhibition with 2 µM TPB-, are also listed in Table I to compare the inhibitory potencies when complete inhibition was achieved with the aid of TPB-.

                              
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Table I
Summary of the inhibition of complex I activity by pyridinium- and quinolinium-type inhibitors
The I75 and I25 are the molar concentrations which gave 75 and 25% inhibition of the control NADH-Q1 oxidoreductase activity without TPB-, respectively. The I50 value, which is the molar concentration to give 50% inhibition, was obtained with 2 µM TPB-. The values are averages from at least two independent measurements.

The dose-response curve of MP-6 (without TPB-) for the inhibition of NADH-Q1 oxidoreductase activity is shown in Fig. 3 (open squares) as a reference. Transformation of a tert-butyl group attached to the benzene ring of MP-6 to an n-butyl group (MP-11, closed circles) or a sec-butyl group (MP-12, closed squares) resulted in reduction of the biphasic nature of the dose-response curve. In particular, saturation of the inhibition by the two derivatives was less clear than that by MP-6. Considering that the hydrophobicities of these three derivatives are almost identical, these findings indicate that a bulky and rigid tert-butyl group of MP-6 is important for the selective inhibition. In the presence of 2 µM TPB-, these three derivatives exhibited similar inhibitory potencies, as shown in the inset in Fig. 3. Complete inhibition was readily attained at concentrations less than 10 µM irrespective of different substituents. The presence of TPB- makes the selective inhibition by pyridinium-type inhibitors ambiguous probably by facilitating inhibitor passage through the hydrophobic barrier to the remaining (i.e. hydrophobic) site (6).


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Fig. 3.   Inhibition of NADH-Q1 oxidoreductase activity by MP-6 and its derivatives. The reaction medium contained 50 mM potassium phosphate (pH 7.4), 0.25 M sucrose, 1 mM MgCl2, 2 mM KCN, 0.4 µM antimycin A, and the final mitochondrial protein concentration was 30 µg/ml. The reaction was started by adding 50 µM NADH after submitochondrial particles were incubated with the indicated concentrations of inhibitors for 4 min at 30 °C. MP-6 (square ), MP-11 (bullet ), and MP-12 (black-square). The inset shows the inhibition by the three inhibitors in the presence of 2 µM TPB-. The control enzyme activity was 0.62 µmol of NADH oxidized per min/mg of protein. The extent of inhibition by 0.1 µM piericidin A was taken as 100% inhibition.

The dose-response curve of the N-ethyl derivative (MP-13) showed no marked biphasic nature, although its inhibitory potency was almost identical to that of MP-6. This result indicates that a methyl group attached to the pyridine nitrogen atom is critical for the selective inhibition. In addition, the observation that the dose-response curves of MP-14 and MP-15 were much less biphasic than that of MP-6 indicates that the para-substitution pattern at the pyridine ring is also important.

Deletion of a branched methyl group in the spacer moiety linking the pyridine and benzene rings (MP-17) and transformation of the methyl to hydroxymethyl (MP-16) results in loss of the biphasic nature of the dose-response curve. These findings demonstrate that the branched methyl group is also an essential structural factor.2

Other derivatives (MP-18 to MP-23) in which the spacer moiety of MP-6 was modified showed no or poorly biphasic titration curves with the exception of MP-23, indicating that three carbon atoms is the best length as the spacer moiety. MP-23, in which the methyl group in MP-6 was replaced by an ethyl group, showed a more significant biphasic nature than that of MP-6 (closed circles in Fig. 4). These results strongly suggest that the branched structure in the spacer moiety is very important to maintain selective inhibition. This is probably because some specific conformation of the two aromatic rings is regulated by steric congestion arising from the branched structure, as discussed below.


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Fig. 4.   Inhibition of NADH-Q1 oxidoreductase activity by MP-6, MP-23, or MP-24. The experimental conditions were the same as those described in the legend to Fig. 3. MP-6 (open circle ), MP-23 (bullet ), or MP-24 (black-square). Inhibition by MP-24 in the presence of 2 µM TPB- (square ) is also shown.

MQ-20, -21, and -22 did not show biphasic dose-response curves, indicating that a quinoline ring cannot substitute for the pyridine ring. The enhancement of their inhibitory potencies by TPB- was slight compared with the corresponding pyridinium compounds (MP-21, MP-22, and MP-6, respectively). This was probably because quinoliniums do not readily form ion pairs with TPB- due to steric hindrance.

Structure/Activity Relationship of Pyridinium-type Inhibitors-- The above results clearly indicated that there are rigid structural constraints for the selective inhibition by MP-6. Namely, a tert-butyl group on the benzene ring, a methyl group attached to the pyridine nitrogen atom, para-substitution in the pyridine ring, and the presence of a branched structure in the spacer moiety are important structural factors for selectivity.

However, the structural factors needed to elicit inhibition per se, not selective, have yet to be defined. Based on the results of structure/activity studies of a wide variety of pyridinium-type inhibitors (6, 10, 17, 21) including the present study, the following conclusions may be drawn: 1) the inhibitory potency increases as the hydrophobicity of the inhibitors increases; 2) a methyl group attached to the pyridine nitrogen atom is not essential because N-ethyl or even bulky N-benzyl derivatives retain the activity; 3) the substitution position as well as the steric shape of the substituents on the pyridinium ring are not restricted; 4) the N-methylpyridinium ring itself is not essential for the activity since other aromatic rings such as bulky N-methylquinolinium can functionally substitute for it. Thus, the physicochemical structural factors, except for hydrophobicity, of the pyridinium-type inhibitors required for inhibition have yet to be defined. It is, however, likely that only two factors, a positive charge (i.e. electrophilic property), which may interact with the proposed anionic residue(s) in the binding site (6), and marked hydrophobicity which facilitates the access of the cationic inhibitor to the binding site through the hydrophobic environment in the membrane, are required for the inhibitory action. We recently showed that the ubiquinone reduction sites of bovine complex I are sufficiently spacious to accommodate exogenous bulky ubiquinone (22). This structural property is extremely unusual compared with other ubiquinone-mediated enzymes such as bovine heart mitochondrial complexes II and III (23), glucose dehydrogenase (24) and terminal ubiquinol oxidases (25) in Escherichia coli. Therefore, the loose recognition by the enzyme of the structure of pyridinium-type inhibitors may reflect the large cavity-like structure of the ubiquinone reduction sites.

Synthetic Development of Novel Selective Inhibitor-- On the basis of the above structure/activity relationship, we synthesized MP-24 which fills the structural requirements for selective inhibition and possesses geminal dimethyl groups in the space moiety. This inhibitor showed a much clearer biphasic dose-response curve (Fig. 4, closed squares) as compared with MP-6 and MP-23. The complete inhibition by MP-24 without TPB- could not be determined because of solubility limit above ~400 µM, but was readily attained at less than 10 µM in the presence of 2 µM TPB- (open squares). The selectivity of MP-24 in terms of the I75/I25 ratio was at least 3-fold clearer than that of MP-6. As discussed for MP-6 (17), the site that is readily blocked by low concentrations of MP-24 without TPB- would be the hydrophilic binding site. The residual enzyme activity in the presence of MP-24 alone was completely inhibited by piericidin A (data not shown). This compound did not entirely inhibit ferricyanide or dichloroindophenol reduction by complex I at concentrations up to ~400 µM, consistent with an earlier report of the original MPP+ (7).

The three-dimensional structure of MP-24 obtained by x-ray crystallographic analysis is shown in Fig. 5.3 This compound is bent at the middle of the molecule due to steric congestion arising from the methyl groups and the aromatic rings. Considering a significant role of the branched structure, the bent conformation must be one of the important structural factors for the selective inhibition.


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Fig. 5.   ORTEP drawing of MP-24.

We confirmed that transformation of para-substitution to ortho-substitution (MP-25) and a tert-butyl group of MP-24 to an n-butyl (MP-26) resulted in marked reduction of selectivity, as observed for pairs of MP-6 versus MP-14 and MP-11, respectively. This structural specificity indicated that selective inhibition is the result of some sort of specific interaction between the inhibitor molecule and the enzyme (or its environment), and exclude the possibility that the apparent saturation of inhibition (i.e. biphasic dose-response curve) was due to artifacts such as solubility limits of the inhibitor.

Transformation of the tert-butyl group of MP-24 to a tert-amyl (MP-27) resulted in slight reduction of the selectivity, indicating that a tert-butyl is the best substituent for this moiety. In addition, MP-28 did not show selective inhibition and its inhibitory potency without TPB- was much poorer than that of MP-24. The inhibitory potency of this compound was not significantly enhanced in the presence of 2 µM TPB-. This was probably because MP-28 does not readily form an ion pair with TPB- due to its bulky and polar branched structure.

Effects of TPB- on the Inhibition by MP-24-- Previously, we reported that in the presence of a small amount of TPB-, the inhibitory potency of some potent MPP+ analogues (such as MQ-17 and MQ-18 in Ref. 17) was markedly enhanced, and complete inhibition was attained at inhibitor concentrations of less than 10 µM (17). In contrast, the presence of an excess amount of TPB- partially reactivated the enzyme activity, and the inhibition was saturated at incomplete level. On the basis of an earlier report (6), we interpreted this partial reversal of inhibition as due to dissociation of the inhibitor from the hydrophilic binding site as a result of an increase in ion-pair formation. However, the inhibitors used previously were not selective inhibitors like MP-24, and variation of TPB- concentrations was not sufficient; therefore, the above hypothesis was re-examined using MP-24 in the presence of various concentrations of TPB-.

Fig. 6 shows the effects of TPB- on the extent of inhibition by MP-24 at two different concentrations (0.9 and 95 µM). At these concentrations, MP-24 exhibited about 30 and 50% inhibition without TPB-, respectively. It is likely that the concentration of 0.9 µM MP-24 was sufficiently low to solely inhibit the hydrophilic site. The extent of inhibition by 0.9 µM MP-24 increased as the concentration of TPB- increased, and maximum inhibition was attained in the presence of approximately equimolar TPB- (closed circles). Further increases in TPB- gradually reactivated the enzyme activity. The inhibition by 95 µM MP-24 was also potentiated by TPB- in a concentration-dependent manner, and complete inhibition was attained in the presence of a catalytic amount of TPB- (closed squares), whereas reactivation was no longer observed. This result supports the previous hypothesis that the presence of an excess amount of TPB- resulted in dissociation of the inhibitor from the hydrophilic binding site.


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Fig. 6.   The effects of TPB- on inhibition of NADH-Q1 oxidoreductase activity by MP-24. The experimental conditions were the same as those described in the legend to Fig. 3. The concentration of MP-24 was 0.9 (bullet ) or 95 µM (black-square). The inhibition by TPB- itself was corrected for the data points of TPB- concentrations above 5 µM.

pH Dependence of the Inhibition by MP-24-- On the basis of pH dependence of the I50 value of MPP+ (plus 10 µM TPB-), Gluck et al. (6) suggested the existence of two distinct ionizable amino acids at or near the MPP+-binding sites. Their experimental conditions, however, were complicated since MPP+ binds simultaneously to both of the sites in the presence of 10 µM TPB- and the inhibition by TPB- itself is unavoidable at this concentration as discussed in the literature (6). We could not reproduce their experimental data (Fig. 5 in Ref. 6), which showed that the I50 value of MPP+ (plus 10 µM TPB-) varies by about 2 orders of magnitude within the pH range of 6-9 with three inflection points.

On the other hand, if the two components of total enzyme activity exhibiting different sensitivities to MP-24 without TPB- (Fig. 4) are indeed attributable to the two distinct binding sites, different pH dependences of the two components would be observed. Therefore, MP-24 might be a suitable inhibitor for the investigation of pH dependence to characterize the properties of the binding sites for pyridinium-type inhibitors. Fig. 7 shows the dose-response curves of MP-24 without TPB- determined at various pH values. The scale of the horizontal axis in the figure differs between the left and right sides. It was clearly shown that inhibitor sensitivity of the high sensitivity component was not affected by the pH changes, whereas that of the low sensitivity component increased markedly when the pH increased over 8.0. This result indicated that MP-24 indeed interacts with the two distinct binding sites in complex I. 


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Fig. 7.   Inhibition of NADH-Q1 oxidoreductase activity by MP-24 under various pH conditions. The experimental conditions were the same as those described in the legend to Fig. 3, except that pH was 6.5 (bullet ), 7.0 (open circle ), 7.5 (black-square), 8.0 (square ), or 8.5 (black-triangle).

Origin of the Selective Inhibition by MP-24-- The marked biphasic nature of the dose-response curve of MP-24 disappeared in the presence of a catalytic amount of TPB-, and complete inhibition was readily attained at low concentrations of the inhibitor (<10 µM) (Fig. 4). Since it is unlikely that MP-24 occupies the binding sites as an ion pair with bulky TPB-, this marked potentiation by TPB- suggested that the energetic barrier preventing the access of positively charged inhibitors to the hydrophobic site is reduced with the aid of TPB- (6, 10). We examined whether this energetic barrier can be overcome by prolongation of incubation time. Fig. 8 shows the dose-response curves of MP-24 determined without TPB- after various incubation periods at 30 °C. It was clear that with longer incubation periods, the biphasic nature became less distinct, indicating that access of MP-24 to the hydrophobic site is promoted by prolongation of incubation time. However, in contrast to the observations in the presence of TPB-, about 15% activity remained even after incubation for 45 min, indicating that complete occupation of the hydrophobic binding site could not be achieved solely by prolongation of incubation time. In our previous study (17), the dose-response curve of MP-6 was not significantly affected by prolongation of incubation time (30 or 120 min). This discrepancy might have been because in the previous study, incubation was performed on ice and then the temperature of the reaction mixture was raised to 30 °C 5 min before starting the enzyme reaction to prevent deactivation of the enzyme. We confirmed that the dose-response curve of MP-24 was not affected by prolongation of incubation time under the previous experimental conditions (data not shown).


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Fig. 8.   Inhibition of NADH-Q1 oxidoreductase activity by MP-24 with various incubation periods. The experimental conditions were the same as those described in the legend to Fig. 3, except that the incubation period was 4 (black-square), 10 (bullet ), 20 (square ), or 45 min (open circle ).

The above results strongly suggest that the process of the inhibitor passage to the binding sites is responsible for the apparently selective inhibition. Since raising the temperature has the same effect as prolongation of the incubation period, we next examined the effects of reaction temperature on the inhibition by MP-24. Fig. 9 shows the dose-response curves of the inhibitor determined at various temperatures with 4 min incubation without TPB- (open symbols). Unexpectedly, the relative inhibition was saturated at ~35, ~20, and ~10% at 25, 20, and 15 °C, respectively. We confirmed that this was not due to insolubility of the inhibitor, and the residual enzyme activity was completely inhibited by 0.1 µM piericidin A (data not shown). In contrast to the observations at 30 °C in which complete inhibition by MP-24 was readily attained at less than 10 µM in the presence of 2 µM TPB- (Fig. 4), the presence of TPB- no longer facilitated complete inhibition at 15 or 20 °C (closed squares and circles, respectively). Complete inhibition by MP-24 with TPB- was achieved at around 200 µM at 25 °C (closed triangles). These results suggested that there is a significant energetic barrier preventing access of the inhibitor to the hydrophilic site as well as the hydrophobic site, although the latter is greater than the former.


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Fig. 9.   Inhibition of NADH-Q1 oxidoreductase activity by MP-24 under various incubation temperatures. The experimental conditions were the same as those described in the legend to Fig. 3, except that the incubation temperature was 15 (square , black-square), 20 (open circle , bullet ), and 25 °C (triangle , black-triangle). Inhibition was determined in the presence (closed symbols) or absence (open symbols) of 2 µM TPB-. As a reference, the titration obtained at 30 °C without TPB- was shown by +.

On the other hand, the I50 values with 2 µM TPB- were almost identical between closely related derivatives such as MP-6, MP-11, and MP-12, and MP-24 and MP-26, although their selectivities in terms of the I75/I25 ratio were entirely different (Table I). This excludes the possibility that a difference in the intrinsic inhibitor sensitivities between the two binding sites was responsible for the observed selective inhibition. This is probably because the inhibitor structure is not strictly recognized by the binding sites, as discussed above. Thus, it is likely that the structural specificity is closely related to the level of the energetic barrier preventing access of the inhibitor to the hydrophobic site. The molecular basis of how the structural specificity is concerned in ease of inhibitor passage is unclear because of the limited available information on three-dimensional structure of the enzyme. Nevertheless, as the sensitivity of the hydrophobic site to the inhibition by MP-24 was significantly enhanced as the pH increased, it is likely that a positively charged amino residue(s) with pKa of around neutral pH (probably histidine, cf. Ref. 6) interferes with access of the inhibitor to the hydrophobic site. Neutralization of the residue(s) by an increase in pH may reduce the energetic barrier of this kinetic process.

    FOOTNOTES

* This work was supported in part by Grant-in-Aid Scientific Research 08660136 (to H. M.) from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Fax: 81-75-753-6408; E-mail: miyoshi{at}kais.kyoto-u.ac.jp.

1 The abbreviations used are: complex I, mitochondrial NADH-ubiquinone oxidoreductase; MPP+, N-methyl-4-phenylpyridinium; Q1, ubiquinone-1; THF, tetrahydrofurane; TPB-, tetraphenylboron.

2 It is necessary to consider the effects of the configuration of the methyl group on the activity since MP-6 is a racemic mixture. However, we did not stereoselectively prepare R and S isomers of MP-6 since this compound is not necessarily the best inhibitor for our purposes.

3 X-ray data for iodine salt of MP-24: Triclinic, space group P-1 (2), a = 12.686(1) Å, b = 14.5214(8) Å, c = 6.1003(4) Å, alpha  = 93.859(6)°, beta  = 100.262(7)°, gamma  = 107.179(5)°, Z = 2, Dcalc = 1.342 g/cm3. Reflection data were collected on a Rigaku AFC5R diffractometer with graphite-monochromated Cu-Kalpha to 2theta max 120.1°; an empirical absorption correction based on azimuthal scans of several reflections was applied. A final refinement gave R(Rw) = 0.029 (0.042) for 2314 refractions with I>3sigma (I). Atomic coordinates have been deposited with the Cambridge Crystallgraphic Data Center.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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