Development of [Ile40]HTLV‐I protease inhibition assay using novel fluorogenic and chromogenic substrate |
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Authors: | Henri‐Obadja Kumada Jeffrey‐Tri Nguyen Taeko Kakizawa Koushi Hidaka Tooru Kimura Yoshio Hayashi Yoshiaki Kiso |
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Affiliation: | 1. Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina‐ku, Kyoto 607‐8412, Japan;2. Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192‐0392, Japan |
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Abstract: | HTLV‐I is a debilitating and/or lethal retrovirus that causes HTLV‐I‐associated myelopathy/tropical spastic paraparesis, adult T‐cell leukemia and several inflammatory diseases. HTLV‐I protease is an aspartic retropepsin involved in HTLV‐I replication and its inhibition could treatHTLV‐I infection. A recombinant L40I mutant HTLV‐I protease was designed and obtained from Escherichia coli, self‐processingand purification by ion‐exchange chromatography. The protease was refolded by a one‐step dialysis and recovered activity. The cleavage efficiency of the [Ile40]HTLV‐I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His‐tagged non‐mutated HTLV‐I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p‐nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV‐I protease inhibition potency assay. The HTLV‐I protease inhibition assay with the [Ile40]HTLV‐I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost‐effective and more time‐efficient while being reproducible and less labor‐intensive. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. |
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Keywords: | human T‐cell lymphotropic virus HTLV‐I‐associated myelopathy/tropical spastic paraparesis adult T‐cell leukemia aspartic protease inhibitor substrate |
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