title: Development of [Ile⁴⁰]HTLV-I protease inhibition assay using novel fluorogenic and chromogenic substrate.
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.
authors.affiliation: Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan.
authors.forename: Henri-Obadja
authors.initials: HO
authors.lastname: Kumada
authors.forename: Jeffrey-Tri
authors.initials: JT
authors.lastname: Nguyen
authors.forename: Taeko
authors.initials: T
authors.lastname: Kakizawa
authors.forename: Koushi
authors.initials: K
authors.lastname: Hidaka
authors.forename: Tooru
authors.initials: T
authors.lastname: Kimura
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002863:Chromogenic Compounds / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D057075:Enzyme Assays / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D015368:Human T-lymphotropic virus 1 / Q000187:drug effects / Q000201:enzymology*; D007532:Isoleucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D007532:Isoleucine; D016282:Aspartic Acid Endopeptidases; C079711:HTLV-1 protease