Testing three different compounds as potential anti-inflammatory drugs for the modulation of LTA4H AP activity

Abstract

Leukotriene A4 hydrolase (LTA4H) is an enzyme affecting neutrophil infiltration in inflammatory diseases like tuberculosis, chronic obstructive pulmonary disease (COPD) and atherosclerosis. LTA4H has two enzymatic pathways: the pro-inflammatory epoxide hydrolase (EH) pathway, where it converts LTA4 to LTB4, and the anti-inflammatory aminopeptidase (AP) pathway, where it cleaves proline-glycine-proline (PGP), the natural substrate of LTA4H, to proline and glycine-proline. Most studies have focused on inhibiting LTA4H’s EH pathway, but fewer studies have focused on promoting its AP pathway. Studies on LTA4H’s AP pathway have mainly been conducted using Ala-pNA as the reporter group because PGP does not have intrinsic fluorescence or absorbance for detection and therefore does not allow for continuous measurements. Herein, we use Pro-pNA as the reporter group, which is more similar to the endogenous PGP structure. For this study, we aimed to determine whether three compounds (B08-2a, B08-2b, and batatasin III), synthesized in our labs, would modulate LTA4H’s AP activity. For the assay, 4-methoxydiphenylmethane (4MDM) and bestatin were used as positive controls for activation or inhibition, respectively. The negative control was treated with buffer alone. Pro-pNA was used as our reporter group. We first produced and purified our LTA4H protein, characterized by a gel electrophoresis, then treated the protein with escalating concentrations of each compound and measured the rate of Pro-pNA cleavage, which was continuously monitored at 405 nm. After analyzing the data, we concluded that B08-2a, B08-2b, and batatasin III inhibit the AP activity with IC50 values of 50.52 μM, 177.68 μM, and 539.04 μM, respectively. More careful assessment and mechanistic characterization of LTA4H enzymatic activities are being pursued to further inform strategies for developing therapeutics for this target. Our next steps will be to test new compounds in order to determine whether any of them will be activators.