As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase (hMPO) offers an attractive protein target to modulate the inflammation of the host tissue through suitable inhibitors. We describe a novel methodology of using low temperature ESR spectroscopy (6 K) and FAST™ technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO. Our initial efforts to profile molecules on the inhibition of MPO-initiated nitration of the Apo-A1 peptide (AEYHAKATEHL) assay showed several potent (with sub-micro molar IC50s) but spurious inhibitors that either do not bind to the heme pocket in the enzyme or retain high (>50 %) anti oxidant potential. Such molecules when taken forward for X-ray did not yield inhibitor-bound co-crystals. We then used ESR to confirm direct binding to the native state enzyme, by measuring the binding-induced shift in the electronic parameter g to rank order the molecules. Molecules with a higher rank order—those with g-shift Rrelative ≥15—yielded well-formed protein-bound crystals (n = 33 structures). The co-crystal structure with the LSN217331 inhibitor reveals that the chlorophenyl group projects away from the heme along the edges of the Phe366 and Phe407 side chain phenyl rings thereby sterically restricting the access to the heme by the substrates like H2O2. Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). In conclusion, our results point to a viable path forward to target the native state of MPO to tame local inflammation.

中文翻译：

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低分子量片段与髓过氧化物酶天然状态的结合及抑制机制的ESR和X射线结构研究


作为受伤位点的早期访客，中性粒细胞来源的人髓过氧化物酶 (hMPO) 提供了一个有吸引力的蛋白质靶标，可通过合适的抑制剂调节宿主组织的炎症。我们描述了一种使用低温 ESR 光谱 (6 K) 和 FAST™ 技术来筛选各种小分子的新方法，这些小分子通过与 MPO 的天然状态可逆结合来抑制过氧化物酶功能。我们最初努力分析分子对 MPO 引发的 Apo-A1 肽硝化的抑制 (AEYHAKATEHL) 测定，结果显示几种有效的（具有亚微摩尔 IC50）但虚假的抑制剂，它们不与酶中的血红素口袋结合或保留高（>50%）抗氧化潜力。当进行 X 射线检测时，此类分子不会产生结合抑制剂的共晶。然后，我们使用 ESR 来确认与天然状态酶的直接结合，通过测量结合诱导的电子参数 g 的变化来对分子进行排序。具有较高等级顺序的分子（g 位移 Rrelative ≥15 的分子）产生形状良好的蛋白质结合晶体（n = 33 个结构）。与 LSN217331 抑制剂的共晶结构表明，氯苯基沿着 Phe366 和 Phe407 侧链苯环的边缘远离血红素，从而在空间上限制 H2O2 等底物接触血红素。 ESR 和抗氧化剂筛选均用于推导作用机制（可逆性、竞争性底物抑制和抗氧化潜力百分比）。总之，我们的结果指出了一条以 MPO 的天然状态为目标来抑制局部炎症的可行途径。