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Potent inhibition of dinuclear zinc(II) peptidase, an aminopeptidase from Aeromonas proteolytica, by 8-quinolinol derivatives: inhibitor design based on Zn2+ fluorophores, kinetic, and X-ray crystallographic study

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Abstract

The selective inhibition of an aminopeptidase from Aeromonas proteolytica (AAP), a dinuclear Zn2+ hydrolase, by 8-quinolinol (8-hydroxyquinoline, 8-HQ) derivatives is reported. We previously reported on the preparation of 8-HQ-pendant cyclens as Zn2+ fluorophores (cyclen is 1,4,7,10-tetraazacyclododecane), in which the nitrogen and phenolate of the 8-HQ units (as well as the four nitrogens of cyclen) bind to Zn2+ in a bidentate manner to form very stable Zn2+ complexes at neutral pH (K d = 8–50 fM at pH 7.4). On the basis of this finding, it was hypothesized that 8-HQ derivatives have the potential to function as specific inhibitors of Zn2+ enzymes, especially dinuclear Zn2+ hydrolases. Assays of 8-HQ derivatives as inhibitors were performed against commercially available dinuclear Zn2+ enzymes such as AAP and alkaline phosphatase. 8-HQ and the 5-substituted 8-HQ derivatives were found to be competitive inhibitors of AAP with inhibition constants of 0.16–29 μM at pH 8.0. The nitrogen at the 1-position and the hydroxide at the 8-position of 8-HQ were found to be essential for the inhibition of AAP. Fluorescence titrations of these drugs with AAP and an X-ray crystal structure analysis of an AAP–8-HQ complex (1.3-Å resolution) confirmed that 8-HQ binds to AAP in the “Pyr-out” mode, in which the hydroxide anion of 8-HQ bridges two Zn2+ ions (Zn1 and Zn2) in the active site of AAP and the nitrogen atom of 8-HQ coordinates to Zn1 (Protein Data Bank code 3VH9).

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Notes

  1. We conducted a potentiometric pH titration of a mixture of 11 and Zn2+–cyclen in aqueous solution with I = 0.1 (NaNO3) at 25 °C to check the complexation of 11 with Zn2+–cyclen. Analysis of the potentiometric pH titration curves suggested little interaction between them at micromolar order concentrations.

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Acknowledgments

This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture in Japan (nos. 19659026, 22390005, and 22659005 for S.A.) and the Academic Frontier project for private universities: matching fund subsidy from MEXT, 2009–2013. We thank Kouya Kobayashi and Yuki Mizuseda for synthesis of some inhibitors and enzyme inhibition assays. The high-performance computing resource was provided by Tokyo University of Science. The synchrotron radiation experiments were performed at BL26B2 at SPring-8 with the approval of RIKEN (proposal no. 20110094) and at BL5A at Photon Factory with the approval of the Photon Factory Program Advisory Committee (proposal no. 2009G660). We thank Masaki Yamamoto and Go Ueno for helpful advice and the coordination of the synchrotron radiation experiments, and we gratefully acknowledge the support of the beamline staff. The high performance computing resource was provided by Tokyo University of Science.

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, 278-8510, Japan

    Kengo Hanaya, Miho Suetsugu & Shin Aoki

  2. Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, 278-8510, Japan

    Shinya Saijo & Ichiro Yamato

  3. RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo, 679-5148, Japan

    Shinya Saijo

  4. Center for Technologies against Cancer, Tokyo University of Science, 2641 Yamazaki, Noda, 278-8510, Japan

    Shin Aoki

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  1. Kengo Hanaya
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Correspondence to Shin Aoki.

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Hanaya, K., Suetsugu, M., Saijo, S. et al. Potent inhibition of dinuclear zinc(II) peptidase, an aminopeptidase from Aeromonas proteolytica, by 8-quinolinol derivatives: inhibitor design based on Zn2+ fluorophores, kinetic, and X-ray crystallographic study. J Biol Inorg Chem 17, 517–529 (2012). https://doi.org/10.1007/s00775-012-0873-4

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