INHIBITORS OF THE MAJOR CYSTEINYL PROTEINASE (GP57/51) IMPAIR HOST-CELL INVASION AND ARREST THE INTRACELLULAR DEVELOPMENT OF TRYPANOSOMA-CRUZI INVITRO
INHIBITORS OF THE MAJOR CYSTEINYL PROTEINASE (GP57/51) IMPAIR HOST-CELL INVASION AND ARREST THE INTRACELLULAR DEVELOPMENT OF TRYPANOSOMA-CRUZI INVITRO
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DOI：
10.1016/0166-6851(92)90050-t
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发表时间：
1992-06-01
影响因子：
1.5
通讯作者：
SCHARFSTEIN, J
中科院分区：
文献类型：
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作者：
MEIRELLES, MNL;JULIANO, L;SCHARFSTEIN, J
肽基重氮甲烷（PDAM）衍生物，一类半胱氨酸蛋白酶的不可逆抑制剂，筛选的能力，损害锥虫克氏锥虫入侵和心肌细胞（HMC）的原代培养细胞内的发展。T. cruzi GP 57/51（一种纯化的半胱氨酰蛋白酶）和底物Z-Phe-Arg-NHMec用于通过连续动力学测定来确定抑制速率常数（k ′ +2）。k '+2值范围为25400 ~ 2800。GP 57/51的最佳抑制剂在P1位置（除了P2之外）具有庞大的疏水残基，因此该酶的S1亚位点特异性类似于哺乳动物组织蛋白酶L。然后研究这些PDAM对寄生虫感染性的影响。当锥虫鞭毛虫短暂暴露于10 μ M的Z-（S-Bzl）Cys-Phe-CHN 2时，入侵HMC的能力明显受损。当PDAM被添加到HMC培养物中时，观察到显著的效果，所述HMC培养物先前已经被锥鞭毛体感染：Z-（S-Bzl）Cys-Phe-CHN，具有0.4 μ M的IC 50，以及不太显著的Z-Phe-Phe-CHN，和Z-Tyr-Phe-CHN 2（或Z-Phe-Tyr-CHN 2）阻断无鞭毛体复制以及它们向锥鞭毛体的转化，从而阻止细胞内发育。相反，Bz-Phe-Gly-CHN 2未能显示抗寄生虫活性。通过用Z-[I-125]Tyr-Phe-CHN 2孵育活的无鞭毛体或感染的HMC，寻求靶半胱氨酸蛋白酶的直接表征。亲和标记暗示GP 57/51作为该探针的主要半胱氨酰蛋白酶靶标。我们建议T. cruzi细胞内发育严重依赖于GP 57/51（cruzipain）。这种半胱氨酰蛋白酶的选择性抑制剂可能具有治疗潜力。
Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. The k'+2 values ranged from 25 400 to 2 800. The best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. The effects of these PDAM on parasite infectivity were then investigated. The ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10-mu-M of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN, with an IC50 of 0.4-mu-M, and less markedly Z-Phe-Phe-CHN, and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[I-125]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.