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A Time-Resolved Fluorescence Probe for Dipeptidyl Peptidase 4 and Its Application in Inhibitor Screening

Authors

  • Mitsuyasu Kawaguchi,

    1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan), Fax: (+81) 3-5841-4855
    2. CREST (Japan) Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan)
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  • Dr. Takayoshi Okabe,

    1. Chemical Biology Research Initiative, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo (Japan)
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  • Takuya Terai,

    1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan), Fax: (+81) 3-5841-4855
    2. CREST (Japan) Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan)
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  • Dr. Kenjiro Hanaoka,

    1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan), Fax: (+81) 3-5841-4855
    2. CREST (Japan) Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan)
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  • Dr. Hirotatsu Kojima,

    1. CREST (Japan) Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan)
    2. Chemical Biology Research Initiative, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo (Japan)
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  • Izumi Minegishi,

    1. Chemical Biology Research Initiative, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo (Japan)
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  • Prof. Dr. Tetsuo Nagano

    Corresponding author
    1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan), Fax: (+81) 3-5841-4855
    2. CREST (Japan) Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan)
    3. Chemical Biology Research Initiative, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo (Japan)
    • Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan), Fax: (+81) 3-5841-4855
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Abstract

The prevalence of type 2 diabetes is increasing dramatically throughout the world. Recently, dipeptidyl peptidase 4 (DPP4) was identified as a potential antidiabetes target. Many DPP4 inhibitors, such as sitagliptin and vildagliptin, have been developed and marketed, but superior therapeutic agents are still required. Therefore, we have developed new methodology for screening of DPP4 inhibitors. Absorption-based measurements with para-nitroaniline or fluorescence-based measurements with the coumarin derivative 7-amino-4-methylcoumarin are often used for the screening of protease inhibitors, including DPP4 inhibitors, but these strategies are not sufficiently sensitive because of interfering background absorption and fluorescence, thus giving rise to many false-positive and false-negative results. Therefore, we have designed and synthesised a novel DPP4 probe (Gly-Pro-BCD-Tb; Gly=glycine, Pro=proline, andBCD defines the backbone of the probe comprising an aniline derivative as on/off switch, a 7-amino-4-methyl-2(1H)-quinolinone (cs-124) as antenna moiety, and a diethylenetriamine-N,N,N’,N’’,N’’-pentaacetic acid (DTPA) as chelator moiety, Tb=terbium) for time-resolved fluorescence (TRF) measurements. TRF measurements with Gly-Pro-BCD-Tb showed high sensitivity and reliability in the inhibitory assay relative to Gly-Pro-MCA (MCA=4-methylcoumarin-7-amide), a conventional fluorescence probe for DPP4. Further, we employed our probe for high-throughput DPP4 inhibitor screening with 3841 randomly selected compounds and found that epibestatin, an epimer of bestatin (a well-known anticancer drug and general aminopeptidase inhibitor), showed dose-dependent DPP4 inhibitory activity. Interestingly, bestatin did not exhibit DPP4 inhibitory activity. We believe that this screening system will be useful for the discovery of DPP4 inhibitors with novel structural scaffolds.

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