Effect of Modifier Structure on the Activation of Leukotriene A₄ Hydrolase Aminopeptidase Activity

Abstract

Activation of the leukotriene A4 hydrolase (LTA4H) aminopeptidase (AP) activity with 4-methoxydiphenylmethane (4MDM) promoted resolution of neutrophil infiltration in a murine cigarette smoke-induced model for emphysematous chronic obstructive pulmonary disease. Recently, 4-(4-benzylphenyl)­thiazol-2-amine (ARM1) was published as a ligand for LTA4H with potential anti-inflammatory properties. To investigate the effect of modifier structure on enzyme kinetics of LTA4H, a series of analogues bearing structural features of ARM1 and 4MDM were synthesized using trifluoroborate Suzuki coupling reactions. Following, the 2.8 Å X-ray crystal structure of LTA4H complexed with 4-OMe-ARM1, a 4MDM-ARM1 hybrid molecule, was determined. Kinetic analysis showed that ARM1 and related analogues lowered affinity for the enzyme–substrate complex, resulting in a change of mechanism from hyperbolic mixed predominately catalytic activation (HMx­(Sp < Ca)­A) as observed for 4MDM to a predominately specific activation (HMx­(Sp > Ca)­A) mechanism. 4-OMe-ARM1 was then shown to dose responsively reduce LTB4 production in human neutrophils.

Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation

“…In the published LTA 4 H:ARM1:OPB-Pro structure (PDB ID: 4MKT) 27 , two rotamers were observed for the side chain of Q136, while the side chain of Q136 in the LTA 4 H:4MDM:OPB-Pro structure showed only one orientation, where the side chain orients toward the catalytic Zn 2+ cation. Also, the crystal structure of LTA 4 H:4MDM showed that Q136 was oriented towards the Zn 2+ cation, which has the same orientation shown in the structure of LTA 4 H:4-OMe-ARM1 complex (PDB ID: 6O5H) 25 . This additional structural information provided by the LTA 4 H:4MDM and LTA 4 H:4MDM:OPB-Pro complexes indicates that Q136 could have a role in tripeptide substrate recognition and catalytic turnover by maintaining rotational freedom 28 .…”

“…Given the high α value for hydrolysis of Arg- p NA in the presence of 4MDM, 4MDM and Arg- p NA cannot simultaneously bind to LTA 4 H, presumably due to the large size of the Arg side chain. However, 4MDM predominantly activates Ala- p NA hydrolysis by increasing catalytic turnover (β > 1) for the EXS complex 25 . In contrast to Arg- and Ala- p NA mechanisms, 4MDM "uncompetitively" activates Pro- p NA hydrolysis where the equilibrium coupling constant α equals the kinetic catalytic constant β (α = β).…”

“…These observations merit attention, because most previous methods seem incomplete and premature to determine even the simple therapeutic effects based on biochemical enzymatic activities. Development of any compounds targeting LTA 4 H will require complete enzymatic characterization as we have done in our recently published work 25 . Moreover, a single therapeutic agent such as 4MDM may experience substrate-dependent mechanisms, leading to highly divergent interactions (such as inhibition or activation) with LTA 4 H. This is clearly an underappreciated aspect of LTA 4 H biology.…”

Particulate matter (PM10) induces in vitro activation of human neutrophils, and lung histopathological alterations in a mouse model

“…Aberrant accumulation and altered functions of neutrophils in the lung are related to increased inflammation and tissue damage in people exposed to PM 26 . In this regard, it has been shown that neutrophils activated by PM exposure are accumulated in the pulmonary vasculature and induce an elevated release of inflammatory mediators such as myeloperoxidase (MPO) 27 and leukotriene B4 (LTB 4 ) 28 . Similarly, an in vitro study showed that neutrophil exposure to PM increased the release of (LTB 4 ), leukotriene C4 (LTC 4 ), and IL-8 29 .…”

IRAP Inhibitors: M1-Aminopeptidase Family Inspiration

“…Clinical trial results to date have been disappointing which has led to the postulate that the inhibiting Pro-Gly-Pro degradation was countering the desired therapeutic effects. In response, the concept of biased inhibitors that spare the aminopeptidase activity has emerged including allosteric ligands (22) that activate Pro-Gly-Pro hydrolysis only (Lee K. H. et al, 2019) or those that block LTA 4 hydrolysis and activate Pro-Gly-Pro hydrolysis.…”