Synthesis and characterization of new piperazine-type inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I).
The mode of action of Deltalac-acetogenins, strong inhibitors of bovine heart mitochondrial complex I, is different from that of traditional inhibitors such as rotenone and piericidin A [Murai, M., et al. (2007) Biochemistry 46 , 6409-6416]. As further exploration of these unique inhibitors might provide new insights into the terminal electron transfer step of complex I, we drastically modified the structure of Deltalac-acetogenins and characterized their inhibitory action. In particular, on the basis of structural similarity between the bis-THF and the piperazine rings, we here synthesized a series of piperazine derivatives. Some of the derivatives exhibited very potent inhibition at nanomolar levels. The hydrophobicity of the side chains and their balance were important structural factors for the inhibition, as is the case for the original Deltalac-acetogenins. However, unlike in the case of the original Deltalac-acetogenins, (i) the presence of two hydroxy groups is not crucial for the activity, (ii) the level of superoxide production induced by the piperazines is relatively high, (iii) the inhibitory potency for the reverse electron transfer is remarkably weaker than that for the forward event, and (iv) the piperazines efficiently suppressed the specific binding of a photoaffinity probe of natural-type acetogenins ([ (125)I]TDA) to the ND1 subunit. We therefore conclude that the action mechanism of the piperazine series differs from that of the original Deltalac-acetogenins. The photoaffinity labeling study using a newly synthesized photoreactive piperazine ([ (125)I]AFP) revealed that this compound binds to the 49 kDa subunit and an unidentified subunit, not ND1, with a frequency of approximately 1:3. A variety of traditional complex I inhibitors as well as Deltalac-acetogenins suppressed the specific binding of [ (125)I]AFP to the subunits. The apparent competitive behavior of inhibitors that seem to bind to different sites may be due to structural changes at the binding site, rather than occupying the same site. The meaning of the occurrence of diverse inhibitors exhibiting different mechanisms of action is discussed in light of the functionality of the membrane arm of complex I.
Ichimaru
Naoya
N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Murai
Masatoshi
M
Kakutani
Nobuyuki
N
Kako
Junko
J
Ishihara
Atsushi
A
Nakagawa
Yoshiaki
Y
Nishioka
Takaaki
T
Yagi
Takao
T
Miyoshi
Hideto
H
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D015394:Molecular Structure; D000077489:Piperazine; D010879:Piperazines / Q000737:chemistry*; D013481:Superoxides / Q000378:metabolism
D004791:Enzyme Inhibitors; D010879:Piperazines; D013481:Superoxides; D000077489:Piperazine; D042967:Electron Transport Complex I
J Biol Chem. 2006 Oct 27;281(43):32724-7
16950771
J Bioenerg Biomembr. 2002 Jun;34(3):193-208
12171069
J Biol Chem. 1994 Aug 19;269(33):21037-42
8063722
Biochemistry. 2005 Nov 15;44(45):14898-906
16274237
J Biol Chem. 2003 Oct 31;278(44):43114-20
12923180
J Biol Chem. 2002 May 17;277(20):17970-7
11880370
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4149-53
10097178
Science. 2006 Mar 10;311(5766):1430-6
16469879
Methods Enzymol. 1984;105:429-35
6328196
J Biol Chem. 1997 Jun 27;272(26):16176-83
9195916
Methods Enzymol. 1983;96:111-20
6361451
Biochemistry. 2003 Jan 28;42(3):746-54
12534287
Biochemistry. 1992 Nov 24;31(46):11413-9
1445878
Biochemistry. 2003 Mar 4;42(8):2266-74
12600193
Eur J Biochem. 2000 May;267(9):2538-46
10785373
Biochemistry. 2005 Jan 18;44(2):816-25
15641810
Q Rev Biophys. 1992 Aug;25(3):253-324
1470679
J Biol Chem. 2004 May 28;279(22):23830-6
15037611
Biochim Biophys Acta. 1998 May 6;1364(2):236-44
9593914
J Neurochem. 2002 Mar;80(5):780-7
11948241
Nature. 1970 Aug 15;227(5259):680-5
5432063
Annu Rev Biochem. 2006;75:69-92
16756485
Biochem Soc Trans. 2005 Jun;33(Pt 3):525-9
15916556
Biochemistry. 2000 Jun 20;39(24):7229-35
10852722
Anal Biochem. 1991 Dec;199(2):223-31
1812789
Biochem J. 1984 Dec 1;224(2):525-34
6517863
Biochemistry. 2006 Aug 15;45(32):9778-87
16893179
Biochim Biophys Acta. 1998 May 6;1364(2):222-35
9593904
J Biol Chem. 2007 Oct 5;282(40):29514-20
17681940
J Biol Chem. 1999 Jan 29;274(5):2625-30
9915790
J Biol Chem. 2003 Jul 11;278(28):25731-7
12730198
IUBMB Life. 2001 Sep-Nov;52(3-5):129-34
11798024
FEBS Lett. 1987 Jul 13;219(1):108-12
3297786
J Biol Chem. 1994 Feb 4;269(5):3167-74
8106350
Eur J Biochem. 1994 Jan 15;219(1-2):691-8
8307034
FEBS Lett. 2001 Nov 23;508(3):385-8
11728457
Biochem J. 2004 Sep 1;382(Pt 2):511-7
15175007
Annu Rev Biochem. 1994;63:675-716
7979252
Biochemistry. 2005 Feb 8;44(5):1744-54
15683258
Biochemistry. 2004 Mar 30;43(12):3750-5
15035646
J Biol Chem. 1998 Apr 3;273(14):8094-8
9525911
Mol Cell Proteomics. 2003 Feb;2(2):117-26
12644575
Biochemistry. 2007 May 29;46(21):6409-16
17474759
Biochemistry. 1994 Apr 19;33(15):4571-6
8161512
Biochemistry. 2004 Mar 30;43(12):3651-8
15035635
J Bioenerg Biomembr. 2000 Dec;32(6):609-15
15254374
Bioorg Med Chem Lett. 2006 Jul 1;16(13):3555-8
16621539
J Biol Chem. 2004 Sep 17;279(38):39414-20
15262965
J Biol Chem. 1971 Apr 25;246(8):2346-53
4324213
J Biol Chem. 1985 Nov 25;260(27):11424-7
4055778
J Biol Chem. 1998 Jul 10;273(28):17368-74
9651320
FEBS Lett. 1998 Jul 10;431(1):34-8
9684860
Annu Rev Physiol. 2004;66:689-733
14977419
Biochim Biophys Acta. 1998 Jul 20;1365(3):443-52
9711297