Development of [Ile⁴⁰]HTLV-I protease inhibition assay using novel fluorogenic and chromogenic substrate

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.