Makaryan Vahagn, Kelley Merideth L, Fletcher Breanna, Bolyard Audrey Anna, Aprikyan A Andrew, Dale David C
Department of Medicine, University of Washington, Seattle, Washington, USA.
Severe Chronic Neutropenia International Registry, University of Washington, Seattle, Washington, USA; and.
J Leukoc Biol. 2017 Oct;102(4):1143-1151. doi: 10.1189/jlb.5A1016-445R. Epub 2017 Jul 28.
Mutations in , the gene for neutrophil elastase (NE), a protease expressed early in neutrophil development, are the most frequent cause of cyclic (CyN) and severe congenital neutropenia (SCN). We hypothesized that inhibitors of NE, acting either by directly inhibiting enzymatic activity or as chaperones for the mutant protein, might be effective as therapy for CyN and SCN. We investigated β-lactam-based inhibitors of human NE (Merck Research Laboratories, Kenilworth, NJ, USA), focusing on 1 inhibitor called MK0339, a potent, orally absorbed agent that had been tested in clinical trials and shown to have a favorable safety profile. Because fresh, primary bone marrow cells are rarely available in sufficient quantities for research studies, we used 3 cellular models: patient-derived, induced pluripotent stem cells (iPSCs); HL60 cells transiently expressing mutant NE; and HL60 cells with regulated expression of the mutant enzyme. In all 3 models, the cells expressing the mutant enzyme had reduced survival as measured with annexin V and FACS. Coincubation with the inhibitors, particularly MK0339, promoted cell survival and increased formation of mature neutrophils. These studies suggest that cell-permeable inhibitors of neutrophil elastase show promise as novel therapies for -associated neutropenia.
中性粒细胞弹性蛋白酶（NE）基因发生突变是周期性（CyN）和严重先天性中性粒细胞减少症（SCN）最常见的病因，NE是一种在中性粒细胞发育早期表达的蛋白酶。我们推测，通过直接抑制酶活性或作为突变蛋白伴侣发挥作用的NE抑制剂，可能作为CyN和SCN的有效治疗方法。我们研究了基于β-内酰胺的人NE抑制剂（美国新泽西州肯尼沃思市默克研究实验室），重点关注一种名为MK0339的抑制剂，这是一种强效、口服吸收的药物，已在临床试验中进行了测试，显示出良好的安全性。由于新鲜的原代骨髓细胞数量很少，难以满足研究需要，我们使用了3种细胞模型：患者来源的诱导多能干细胞（iPSC）；瞬时表达突变NE的HL60细胞；以及突变酶表达受调控的HL60细胞。在所有3种模型中，用膜联蛋白V和流式细胞术检测发现，表达突变酶的细胞存活率降低。与抑制剂共同孵育，尤其是MK0339，可促进细胞存活并增加成熟中性粒细胞的形成。这些研究表明，可渗透细胞的中性粒细胞弹性蛋白酶抑制剂有望成为相关中性粒细胞减少症的新型治疗方法。
