Kinetic Characterization of Mitochondrial Complex I Inhibitors Using Annonaceous Acetogenins (original) (raw)
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Bioorganic & Medicinal Chemistry Letters, 2006
We synthesized a series of Dlac-acetogenins in which the two alkyl side chains were systematically modified, and examined their inhibitory effect on bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). The results revealed that the physicochemical properties of the side chains, such as the balance of hydrophobicity and the width (or bulkiness) of the chains, are important structural factors for a potent inhibitory effect of amphiphilic Dlac-acetogenins. This is probably because such properties decide the precise location of the hydrophilic bis-THF ring moiety in the enzyme embedded in the inner mitochondrial membrane.
Current Topics of the Inhibitors of Mitochondrial Complex I
A Structural Perspective on Respiratory Complex I, 2012
Acetogenins isolated from Annonaceous plants are very potent inhibitors of bovine heart mitochondrial complex I and unique in chemical structure among a wide variety of inhibitors of the enzyme. The structure-activity studies of numerous acetogenins revealed that the inhibitors exhibit potent inhibition only when the two pharmacophores (i.e. the g-lactone and the hydroxylated THF moieties) are directly linked by the alkyl spacer and cooperatively bind to the two putative binding sites. Regardless of marked changes in the conformation and/or the length of the spacer region, the spacer dynamically regulates the cooperative binding of the two pharmacophores to the sites. Through structural modi fi cation of acetogenins, we developed new types of inhibitors termed D lac-acetogenins and piperazine-type inhibitors, whose inhibitory effects on complex I signi fi cantly differ from those of traditional inhibitors. In particular, examination of the inhibition mechanism of D lac-acetogenins provided valuable insights into the terminal electron transfer step of complex I including a mechanism of the generation of superoxide in the presence of inhibitor. Using a photoaf fi nity labeling technique, the binding sites of the new inhibitors (D lac-acetogenin and piperazine) as well as traditional inhibitors (acetogenin and quinazoline) were identi fi ed at sub-subunit level. The photoaf fi nity labeling studies clearly indicated that the 49 kDa and ND1 subunits construct a large inhibitor binding pocket in bovine complex I.
Journal of bioenergetics and biomembranes, 2001
Studies of the structure-activity relationships of ubiquinones and specific inhibitors are helpful to probe the structural and functional features of the ubiquinone reduction site of bovine heart mitochondrial complex I. Bulky exogenous short-chain ubiquinones serve as sufficient electron acceptors from the physiological ubiquinone reduction site of bovine complex I. This feature is in marked contrast to other respiratory enzymes such as mitochondrial complexes II and III. For various complex I inhibitors, including the most potent inhibitors, acetogenins, the essential structural factors that markedly affect the inhibitory potency are not necessarily obvious. Thus, the loose recognition by the enzyme of substrate and inhibitor structures may reflect the large cavity like structure of the ubiquinone (or inhibitor) binding domain in the enzyme. On the other hand, several phenomena are difficult to explain by a simple one-catalytic site model for ubiquinone.
Proton pumping of mitochondrial complex I: differential activation by analogs of ubiquinone
Journal of bioenergetics and biomembranes, 1997
As part of the ongoing studies aimed at elucidating the mechanism of the energy conserving function of mitochondrial complex I, NADH: ubiquinone (Q) reductase, we have investigated how short-chain Q analogs activate the proton pumping function of this complex. Using a pH-sensitive fluorescent dye we have monitored both the extent and initial velocity of proton pumping of complex I in submitochondrial particles. The results are consistent with two sites of interaction of Q analogs with complex I, each having different proton pumping capacity. ...
Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
125 I-labeled (trifluoromethyl)phenyldiazirinyl acetogenin, [ 125 I]TDA, a photoaffinity labeling probe of acetogenin, photo-cross-links to the ND1 subunit of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) with high specificity [ M. Murai, A. Ishihara, T. Nishioka, T. Yagi, and H. Miyoshi, (2007) The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I, Biochemistry 46 6409-6416.]. To identify the binding site of [ 125 I]TDA in the ND1 subunit, we carried out limited proteolysis of the subunit cross-linked by [ 125 I]TDA using various proteases and carefully analyzed the fragmentation patterns. Our results revealed that the cross-linked residue is located within the region of the 4th to 5th transmembrane helices (Val144-Glu192) of the subunit. It is worth noting that an excess amount of shortchain ubiquinones such as ubiquinone-2 (Q 2) and 2-azido-Q 2 suppressed the cross-linking by [ 125 I]TDA in a concentration-dependent way. Although the question of whether the binding sites for ubiquinone and different inhibitors in complex I are identical remains to be answered, the present study provided, for the first time, direct evidence that an inhibitor (acetogenin) and ubiquinone competitively bind to the enzyme. Considering the present results along with earlier photoaffinity labeling studies, we propose that not all inhibitors acting at the terminal electron transfer step of complex I necessarily bind to the ubiquinone binding site itself.
Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species
Biochimica Et Biophysica Acta-bioenergetics, 2009
We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2′,7′-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q 2 ) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q 1 as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decylubiquinone. This behaviour relates the prooxidant CoQ 1 activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH-DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron-sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.
2021
ND1 subunit possesses the majority of the inhibitor binding domain of the human mitochondrial respiratory complex I. This is an attractive target for the search for new inhibitors that seek mitochondrial dysfunction. It is known, from in vitro experiments, that some metabolites from Annona muricata called acetogenins have important biological activities, such as anticancer, antiparasitic, and insecticide. Previous studies propose an inhibitory activity of bovine mitochondrial respiratory complex I by bis-tetrahydrofurans acetogenins such as annocatacin B, however, there are few studies on its inhibitory effect on human mitochondrial respiratory complex I. In this work, we evaluate the in silico molecular and energetic affinity of the annocatacin B molecule with the human ND1 subunit in order to elucidate its potential capacity to be a good inhibitor of this subunit. For this purpose, quantum mechanical optimizations, molecular dynamics simulations and the molecular mechanics/Poisson...