title: Synthesis and characterization of new piperazine-type inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I).
abstract: 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.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Ichimaru
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Murai
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kakutani
authors.forename: Junko
authors.initials: J
authors.lastname: Kako
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ishihara
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: Takaaki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
authors.forename: Hideto
authors.initials: H
authors.lastname: Miyoshi
mesh_terms: 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
chemical_list: D004791:Enzyme Inhibitors; D010879:Piperazines; D013481:Superoxides; D000077489:Piperazine; D042967:Electron Transport Complex I
references.citation: J Biol Chem. 2006 Oct 27;281(43):32724-7
references.pmid: 16950771
references.citation: J Bioenerg Biomembr. 2002 Jun;34(3):193-208
references.pmid: 12171069
references.citation: J Biol Chem. 1994 Aug 19;269(33):21037-42
references.pmid: 8063722
references.citation: Biochemistry. 2005 Nov 15;44(45):14898-906
references.pmid: 16274237
references.citation: J Biol Chem. 2003 Oct 31;278(44):43114-20
references.pmid: 12923180
references.citation: J Biol Chem. 2002 May 17;277(20):17970-7
references.pmid: 11880370
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4149-53
references.pmid: 10097178
references.citation: Science. 2006 Mar 10;311(5766):1430-6
references.pmid: 16469879
references.citation: Methods Enzymol. 1984;105:429-35
references.pmid: 6328196
references.citation: J Biol Chem. 1997 Jun 27;272(26):16176-83
references.pmid: 9195916
references.citation: Methods Enzymol. 1983;96:111-20
references.pmid: 6361451
references.citation: Biochemistry. 2003 Jan 28;42(3):746-54
references.pmid: 12534287
references.citation: Biochemistry. 1992 Nov 24;31(46):11413-9
references.pmid: 1445878
references.citation: Biochemistry. 2003 Mar 4;42(8):2266-74
references.pmid: 12600193
references.citation: Eur J Biochem. 2000 May;267(9):2538-46
references.pmid: 10785373
references.citation: Biochemistry. 2005 Jan 18;44(2):816-25
references.pmid: 15641810
references.citation: Q Rev Biophys. 1992 Aug;25(3):253-324
references.pmid: 1470679
references.citation: J Biol Chem. 2004 May 28;279(22):23830-6
references.pmid: 15037611
references.citation: Biochim Biophys Acta. 1998 May 6;1364(2):236-44
references.pmid: 9593914
references.citation: J Neurochem. 2002 Mar;80(5):780-7
references.pmid: 11948241
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Annu Rev Biochem. 2006;75:69-92
references.pmid: 16756485
references.citation: Biochem Soc Trans. 2005 Jun;33(Pt 3):525-9
references.pmid: 15916556
references.citation: Biochemistry. 2000 Jun 20;39(24):7229-35
references.pmid: 10852722
references.citation: Anal Biochem. 1991 Dec;199(2):223-31
references.pmid: 1812789
references.citation: Biochem J. 1984 Dec 1;224(2):525-34
references.pmid: 6517863
references.citation: Biochemistry. 2006 Aug 15;45(32):9778-87
references.pmid: 16893179
references.citation: Biochim Biophys Acta. 1998 May 6;1364(2):222-35
references.pmid: 9593904
references.citation: J Biol Chem. 2007 Oct 5;282(40):29514-20
references.pmid: 17681940
references.citation: J Biol Chem. 1999 Jan 29;274(5):2625-30
references.pmid: 9915790
references.citation: J Biol Chem. 2003 Jul 11;278(28):25731-7
references.pmid: 12730198
references.citation: IUBMB Life. 2001 Sep-Nov;52(3-5):129-34
references.pmid: 11798024
references.citation: FEBS Lett. 1987 Jul 13;219(1):108-12
references.pmid: 3297786
references.citation: J Biol Chem. 1994 Feb 4;269(5):3167-74
references.pmid: 8106350
references.citation: Eur J Biochem. 1994 Jan 15;219(1-2):691-8
references.pmid: 8307034
references.citation: FEBS Lett. 2001 Nov 23;508(3):385-8
references.pmid: 11728457
references.citation: Biochem J. 2004 Sep 1;382(Pt 2):511-7
references.pmid: 15175007
references.citation: Annu Rev Biochem. 1994;63:675-716
references.pmid: 7979252
references.citation: Biochemistry. 2005 Feb 8;44(5):1744-54
references.pmid: 15683258
references.citation: Biochemistry. 2004 Mar 30;43(12):3750-5
references.pmid: 15035646
references.citation: J Biol Chem. 1998 Apr 3;273(14):8094-8
references.pmid: 9525911
references.citation: Mol Cell Proteomics. 2003 Feb;2(2):117-26
references.pmid: 12644575
references.citation: Biochemistry. 2007 May 29;46(21):6409-16
references.pmid: 17474759
references.citation: Biochemistry. 1994 Apr 19;33(15):4571-6
references.pmid: 8161512
references.citation: Biochemistry. 2004 Mar 30;43(12):3651-8
references.pmid: 15035635
references.citation: J Bioenerg Biomembr. 2000 Dec;32(6):609-15
references.pmid: 15254374
references.citation: Bioorg Med Chem Lett. 2006 Jul 1;16(13):3555-8
references.pmid: 16621539
references.citation: J Biol Chem. 2004 Sep 17;279(38):39414-20
references.pmid: 15262965
references.citation: J Biol Chem. 1971 Apr 25;246(8):2346-53
references.pmid: 4324213
references.citation: J Biol Chem. 1985 Nov 25;260(27):11424-7
references.pmid: 4055778
references.citation: J Biol Chem. 1998 Jul 10;273(28):17368-74
references.pmid: 9651320
references.citation: FEBS Lett. 1998 Jul 10;431(1):34-8
references.pmid: 9684860
references.citation: Annu Rev Physiol. 2004;66:689-733
references.pmid: 14977419
references.citation: Biochim Biophys Acta. 1998 Jul 20;1365(3):443-52
references.pmid: 9711297