Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.
Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.
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DOI：
10.1177/1087057114525852
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发表时间：
2014-08
影响因子：
--
通讯作者：
Hodder P
中科院分区：
文献类型：
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作者：
Spicer T;Fernandez-Vega V;Chase P;Scampavia L;To J;Dalton JP;Da Silva FL;Skinner-Adams TS;Gardiner DL;Trenholme KR;Brown CL;Ghosh P;Porubsky P;Wang JL;Whipple DA;Schoenen FJ;Hodder P
这项研究的目标，PfM 18乙酰氨基肽酶（PfM 18 AAP），是唯一的AAP存在于疟疾寄生虫恶性疟原虫的基因组中。PfM 18 AAP是一种金属外肽酶，专门切割N-末端酸性氨基酸谷氨酸和天冬氨酸。它在寄生虫细胞质中表达，并可能与其他氨肽酶一起在蛋白质降解（例如血红蛋白）中发挥作用。先前的反义敲除实验鉴定了与PfM 18 AAP相关的致死表型，表明其是新的抗疟疾疗法的有效靶标。为了鉴定PfM 18 AAP功能的抑制剂，使用重组PfM 18 AAP酶和荧光肽底物（H-Glu-NHMec）开发了荧光酶促测定。这是根据分子库探针生产中心网络（MLPCN）收集的约292，000种化合物（分子库小分子储存库（MLSMR））进行筛选的。开发了一种基于组织蛋白酶L1（CTSL 1）的测定法，并将其用作一种反筛选，以鉴定具有非特异性活性的化合物。酶学和表型测定用于确定从HTS鉴定的选择性和有效化合物的作用机制和功效。两种结构相关的化合物CID 6852389和CID 23724194产生微摩尔效价，在CTSL 1滴定实验中无活性（IC 50>59.6 μM）。如通过Ki测定所测量的，两种化合物在PfM 18 AAP酶测定中显示微摩尔非竞争性抑制。CID 6852389和CID 23724194均在疟疾生长试验中表现出效价（IC 50分别为4 μM和1.3 μM）。
The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP suggesting that it is a valid target for new anti-malaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network (MLPCN) collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository (MLSMR)). A Cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counterscreen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from HTS. Two structurally related compounds, CID 6852389 and CID 23724194, yield micromolar potency and are inactive in CTSL1 titration experiments (IC50 >59.6 μM). As measured by Ki assay, both compounds demonstrate micromolar non-competitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrate potency in malaria growth assays (IC50 4 μM and 1.3 μM, respectively).