Hideaki Ui

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues.

Guadinomines, Type III Secretion System Inhibitors, Produced by Streptomyces sp. K01-0509

Enteropathogenic Escherichia coli (EPEC) expressing the Type III secretion system (TTSS) induced hemolysis of sheep blood cells. Using this assay, six structurally related compounds designated as guadinomines were isolated as inhibitors of TTSS-induced hemolysis by ion exchange column chromatography and HPLC from the culture broth of Streptomyces sp. K01-0509. Guadinomines A and B showed potent inhibition with IC50 values of 0.02 and 0.007 μg/ml, respectively, guadinomine D showed moderate activity (IC50: 8.5 μg/ml), while guadinomines C1 and C2 and guadinomic acid had no activity.

Chitinase inhibitors: extraction of the active framework from natural argifin and use of in situ click chemistry.

In situ click chemistry is a target-guided synthesis technique for discovering potent protein ligands by assembling azides and alkynes into triazoles inside the affinity site of a target protein. We report the rapid discovery of a new and potent inhibitor of bacterial chitinases by the use of in situ click chemistry. We observed a target-templated formation of a potent triazole inhibitor of the chitinase-catalyzed chitin hydrolysis, through in situ click chemistry between a biologically active azide-containing scaffold and structurally unrelated alkyne fragments. Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. Argifin, which has been isolated and characterized as a cyclopentapeptide natural product by our research group, shows strong inhibitory activity against chitinases. As a result of our efforts at developing a chitinase inhibitor from an azide-bearing argifin fragment and the application of the chitinase template and a library of alkynes, we rapidly obtained a very potent and new 1,5-disubstituted triazole inhibitor against Serratia marcescens chitinase (SmChi) B. The new inhibitor expressed 300-fold increase in the inhibitory activity against SmChiB compared with that of argifin. To the best of our knowledge, our finding of an enzyme-made 1,5-disubstituted triazole, using in situ click chemistry is the second example reported in the literature.

Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II).

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans, we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.

Guadinomines, Type III secretion system inhibitors, produced by Streptomyces sp. K01-0509. II: physico-chemical properties and structure elucidation.

The structures of guadinomines, new inhibitors of a bacterial Type III secretion system produced by Streptomyces sp. K01-0509, were elucidated by spectroscopic studies including various NMR experiments. Guadinomines A, B, C1, C2 and D consist of a carbamoylated cyclic guanidinyl moiety, an alkyl chain moiety and an L-Ala-L-Val moiety in common, while guadinomic acid is a smaller molecule consisting of a carbamoylated cyclic guanidinyl moiety and a hydroxyl hexanoate moiety.

Gramicidin S identified as a potent inhibitor for cytochrome bd‐type quinol oxidase

Gramicidin S, a cationic cyclic decapeptide, exhibits the potent antibiotic activity through perturbation of lipid bilayers of the bacterial membrane. From the screening of natural antibiotics, we identified gramicidin S as a potent inhibitor for cytochrome bd‐type quinol oxidase from Escherichia coli. We found that gramicidin S inhibited the oxidase with IC50 of 3.5 μM by decreasing V max and the affinity for substrates but showed the stimulatory effect at low concentrations. Our findings would provide a new insight into the development of gramicidin S analogs, which do not share the target and mechanism with conventional antibiotics.

Absolute configuration of nafuredin, a new specific NADH-fumarate reductase inhibitor

Abstract Nafuredin, a new specific NADH-fumarate reductase inhibitor, was isolated from the culture broth of a fungal strain Aspergillus niger FT-0554. The stereoselective synthesis of three degradation products obtained by ozonolysis of nafuredin allowed elucidation of the absolute configuration of nafuredin.

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272beta, gamma, delta, epsilon and zeta, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272beta and epsilon (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272gamma, delta, and zeta (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272beta and delta, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.

Biochemical and Spectroscopic Properties of Cyanide-Insensitive Quinol Oxidase from Gluconobacter oxydans

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.

Argifin; efficient solid phase total synthesis and evalution of analogues of acyclic peptide

An effective solid phase synthesis of Argifin, providing subsequent access to effective synthesis of analogues, was developed in 13% overall yield, as well as elucidating structure-activity relationships. The novel acyclic peptide 18b, prepared from a synthetic intermediate of Argifin, was found to be 70 times more potent as an inhibitor of Serratia marcescens chitinases B than Argifin itself.