Identification of Bioactivating Enzymes Involved in the Hydrolysis of Laninamivir Octanoate, a Long-Acting Neuraminidase Inhibitor, in Human Pulmonary Tissue

Abstract

Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LOhydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling-a technology integration of traditional biochemistry and proteomics. In a single elution step by gelfiltration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.

Tissue-Specific Proteomics Analysis of Anti-COVID-19 Nucleoside and Nucleotide Prodrug-Activating Enzymes Provides Insights into the Optimization of Prodrug Design and Pharmacotherapy Strategy

“…Laninamivir octanoate is a long-lasting neuraminidase inhibitor for the treatment of influenza . Lysophospholipase 1 (LYPLA1) and esterase D (ESD) were identified as the major enzymes responsible for hydrolysis of laninamivir octanoate in human lung S9 fractions, with catalytic activities of 0.377 and 0.232 pmol/min/μg of enzyme, respectively . The pulmonary expression of ESD was comparable to its renal expression and higher than its intestinal expression, but it was lower than the liver expression.…”

“…Hydrolysis efficiency of laninamivir octanoate in different human tissues. (a) Activation pathway of laninamivir octanoate activation . (b) Protein levels of ESD and LYPLA1.…”

“…Laninamivir octanoate was selected as an example of a long-acting inhaled antiviral prodrug . The activating enzymes and activation mechanisms of these prodrugs were obtained from the literature and carefully reviewed and scrutinized. ,,− …”

“…The S9 fraction/tissue scaling factors were adopted from a previous study . Data concerning the activities of purified recombinant human enzymes were extracted from the literature. ,,,− We used eq to calculate each enzyme’s contribution (contrib.) to the metabolism of a given substrate drug in a specific tissue, with an assumption that the activity of a recombinant (recomb.)…”

Intrapulmonary Pharmacokinetics of Laninamivir, a Neuraminidase Inhibitor, after a Single Nebulized Administration of Laninamivir Octanoate in Healthy Japanese Subjects

“…This prolonged retention of laninamivir in the respiratory tissues was explained by a consecutive series of LO uptake, hydrolysis, and limited efflux of laninamivir in mice (13). Both S-formylglutathione hydrolase (esterase D) and acyl-protein thioesterase 1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue (14). In addition, laninamivir bound to viral neuraminidase more stably in vitro than other neuraminidase inhibitors, including oseltamivir, zanamivir, and peramivir (15).…”