Division of Pulmonary, Allergy and Critical Care, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, United States.
Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, United States.
Biochim Biophys Acta Gen Subj. 2018 Jun;1862(6):1364-1375. doi: 10.1016/j.bbagen.2018.03.003. Epub 2018 Mar 7.
Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils.
MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site.
LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl. EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site.
We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage.
髓过氧化物酶（MPO）在炎症和感染期间生成次氯酸（HOCl）。我们发现，开环异落叶松脂素二葡萄糖苷（SDG）可以清除生理溶液中辐射诱导的 HOCl。然而，SDG 及其合成版本 LGM2605 对 MPO 催化生成 HOCl 的作用尚不清楚。本研究评估了 LGM2605 对人 MPO 以及巨噬细胞和中性粒细胞来源的鼠 MPO 的作用。
使用次氯酸盐特异性 3'-(对-氨基苯)荧光素（APF）通过荧光法测定 MPO 活性。使用 Amplex Red 测定 LGM2605 对 MPO 过氧化物酶循环的影响，同时使用牛磺酸氯胺测定法测定其对氯化循环的影响。使用电子顺磁共振（EPR）光谱学测定 LGM2605 对 MPO 的 EPR 信号的影响。最后，使用计算对接确定 SDG 对酶活性位点的能量有利的对接构象。
LGM2605 抑制人源和鼠源 MPO 活性。在没有 Cl 的情况下以及存在 Cl 的情况下均观察到 MPO 抑制。EPR 证实 LGM2605 抑制了 MPO 催化循环中的 Compound I 的形成，Compound I 是一种含 MPO 过氧化物酶的卟啉 π-自由基的氧化态铁（IV）中间物 [Fe(IV)O]。计算对接表明 SDG 可以通过与酶的活性位点结合而充当抑制剂。
我们得出结论，LGM2605 通过抑制过氧化物酶和氯化循环来抑制 MPO 活性。EPR 分析表明，LGM2605 通过减少高度氧化的 Compound I 的形成来抑制 MPO。本研究确定了 LGM2605 作为 MPO 抑制剂的新作用机制，并表明 LGM2605 可能是一种有前途的氧化剂依赖性炎症性组织损伤抑制剂。
