Activation and Inhibition of Leukotriene A4 Hydrolase Aminopeptidase Activity by Modulators B08-2a and B08-2b

Abstract

: Leukotriene A₄ hydrolase (LTA₄H) is a bi-functional enzyme that promotes a pro-inflammatory epoxy hydrolase (EH) pathway and an anti-inflammatory aminopeptidase (AP) pathway. LTA₄H EH activity catalyzes the hydrolysis of leukotriene A₄ (LTA₄) to the neutrophil chemoattractant leukotriene B₄ (LTB₄), while AP activity hydrolyzes the tripeptide proline-glycine-proline (PGP) to clear neutrophils in the lungs. Previously published studies focused on blocking EH activity to inhibit the LTB₄ formation with small molecule inhibitors. Our research focuses on the activation of LTA₄H AP activity in the presence of small molecule activators.  Prior to kinetic analysis, a standard curve was obtained using para-nitroanilide (pNA) at escalating concentrations. A chromogenic substrate alanine-p-nitroanilide (Ala-pNA) was used to measure the AP activity of LTA₄H. Using the 4-methoxydiphenylmethane (4MDM) as a positive control for activating the AP activity and bestatin as a negative control for inhibiting AP activity, the modulation of LTA₄H AP activity was analyzed in the presence of compounds B08-2a and B08-2b through the determination of a 50% activation or inhibition (AC₅₀/IC₅₀) kinetic assay. In conclusion, our research involves the high-throughput assay for screening small molecules to modulate the AP activity of LTA₄H. The discovery of small molecules that activate the AP activity of the LTA₄H enzyme is a potential new therapeutic agent for lung inflammatory diseases, such as chronic obstructive pulmonary disease (COPD).