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
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 |
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).
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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.
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EXPERIMENTAL PROCEDURES |
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.
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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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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 (
= 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.
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RESULTS AND DISCUSSION |
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.
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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 ( ), MP-11 ( ), and MP-12
( ). 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.
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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 ( ), MP-23 ( ), or MP-24 ( ). Inhibition by MP-24 in the
presence of 2 µM TPB ( ) is also
shown.
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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.
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 ( ) or 95 µM ( ). The inhibition by TPB
itself was corrected for the data points of TPB
concentrations above 5 µM.
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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 ( ), 7.0 ( ), 7.5 ( ), 8.0 ( ), or 8.5 ( ).
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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 ( ), 10 ( ), 20 ( ), or 45 min ( ).
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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 ( , ), 20 ( , ), and 25 °C ( , ).
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 +.
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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.