Biochemical characterization of the inhibition mechanism of Deltalac-acetogenins synthesized in our laboratory indicated that they are a new type of inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) [Murai, M., et al. (2006) Biochemistry 45, 9778-9787]. To identify the binding site of Deltalac-acetogenins with a photoaffinity labeling technique, we synthesized a photoreactive Deltalac-acetogenin ([(125)I]diazinylated Deltalac-acetogenin, [(125)I]DAA) which has a small photoreactive diazirine group attached to a pharmacophore, the bis-THF ring moiety. Characterization of the inhibitory effects of DAA on bovine complex I revealed unique features specific to, though not completely the same as those of, the original Deltalac-acetogenin. Using [(125)I]DAA, we carried out photoaffinity labeling with bovine heart submitochondrial particles. Analysis of the photo-cross-linked protein by Western blotting and immunoprecipitation revealed that [(125)I]DAA binds to the membrane subunit ND1 with high specificity. The photo-cross-linking to the ND1 subunit was suppressed by an exogenous short-chain ubiquinone (Q(2)) in a concentration-dependent manner. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by limited proteolysis using lysylendopeptidase, endoprotease Asp-N, or trypsin and their changes in the presence of the original Deltalac-acetogenin strongly suggested that the cross-linked residues are located at two different sites in the third matrix-side loop connecting the fifth and sixth transmembrane helices.

The mode of action of Δlac-acetogenins, strong inhibitors of bovine heart mitochondrial complex I, is different from that of traditional inhibitors such as rotenone and piericidin A [Murai 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 Δlac-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 Δlac-acetogenins. However, unlike in the case of the original Δlac-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. It is therefore concluded that the action mechanism of the piperazine series differs from that of the original Δlac-acetogenins. 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 about 1:3. A variety of traditional complex I inhibitors as well as Δlac-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 the light of the functionality of the membrane arm of complex I.NADH-ubiquinone oxidoreductase (complex I) 1 is the first energy-transducing enzyme of the respiratory chains of most mitochondria and many bacteria. The enzyme catalyzes the transfer † This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant 17380073 to H. M.), a Grant-in-Aid for JSPS Fellows (to N. I. and M. M.), and NIH grant R01GM033712 (to T. Y.).*To whom correspondence should be addressed: e-mail, miyoshi@kais.kyoto-u.ac.jp; Tel., +81−75−753−6119; Fax, +81−75−753 −6408.. Table S1, and Figures S1-S3. This material is available free of charge via the Internet at http://pubs.acs.org. SUPPORTING INFORMATION AVAILABLE Syntheses of compounds 4−24 and [ 125 I]AFP, NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 October 7. NIH-PA Author Manu...

This study was aimed to predict drug-induced liver injury caused by reactive metabolites. Reactive metabolites covalently bind to proteins and could result in severe outcomes in patients. However, the relation between the extent of covalent binding and clinical hepatotoxicity is still unclear. From a perspective of body burden (human in vivo exposure to reactive metabolites), we developed a risk assessment method in which reactive metabolite burden (RM burden), an index that could reflect the body burden associated with reactive metabolite exposure, is calculated using the extent of covalent binding, clinical dose, and human in vivo clearance. The relationship between RM burden and hepatotoxicity in humans was then investigated. The results indicated that this RM burden assessment exhibited good predictability for sensitivity and specificity, and drugs with over 10 mg/day RM burden have high-risk for hepatotoxicity. Furthermore, a quantitative trapping assay using radiolabeled trapping agents ([ 35 S] cysteine and [ 14 C]KCN) was also developed, to detect reactive metabolite formation in the early drug discovery stage. RM burden calculated using this assay showed as good predictability as RM burden calculated using conventional time-and cost-consuming covalent binding assays. These results indicated that the combination of RM burden and our trapping assay would be a good risk assessment method for reactive metabolites from the drug discovery stage.

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