Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase
Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase
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DOI：
10.1074/jbc.m117.801779
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发表时间：
2017-09-22
影响因子：
4.8
通讯作者：
Schramm, Vern L.
中科院分区：
文献类型：
--
作者：
Gebre, Sara T.;Cameron, Scott A.;Schramm, Vern L.
嘌呤核苷磷酸化酶（PNP）是人类嘌呤补救途径的一部分。它的缺陷会引发活化 T 细胞的凋亡，使其成为 T 细胞增殖性疾病的目标。 PNP 的过渡态类似物以皮摩尔 (pM) 解离常数结合。紧密结合的 PNP 抑制剂对目标酶表现出极长的寿命。我们通过比较体外和体内的功能解离速率来解决目标停留时间的机制。我们报告了 7 种 Immucillins 的体外 PNPinhibitor 解离率 (t(1/2))，从 3 到 31 分钟，解离常数为 115 到 6 pM。用 DADMe-Immucillin-H (DADMe-ImmH, 22 pM) 处理人红细胞会导致 PNP 完全抑制。在多次洗涤过程中，[C-14] DADMe-ImmH 从红细胞中的损失是缓慢且双相的，这是由抑制剂释放和重新结合到 PNP 催化位点造成的。慢相的 t(1/2) 为 84 小时。在存在过量未标记的 DADMe-ImmH 的情况下，红细胞中 [C-14] DADMe-ImmH 的损失通过防止重新结合而增加至 1.6 小时的 t(1/2)。因此，在人红细胞中，DADMe-ImmH重新结合的可能性比红细胞中抑制剂扩散损失的可能性高50倍。在 1 期临床试验中，接受单次口服剂量 DADMe-ImmH 治疗的人类在 t(1/2) 为 59 天时表现出 PNP 活性的恢复，与人类的红细胞生成率相对应。因此，DADMe-ImmH 的 PNP 催化位点重新捕获在体内受到高度青睐。我们得出的结论是，具有皮摩尔解离常数的过渡态类似物在体内的靶标上表现出较长的寿命，因为靶标酶重新捕获抑制剂分子的概率大于扩散损失到细胞外空间的概率。
Purine nucleoside phosphorylase (PNP) is part of the human purine salvage pathway. Its deficiency triggers apoptosis of activated T-cells, making it a target for T-cell proliferative disorders. Transition-state analogues of PNP bind with picomolar (pM) dissociation constants. Tight-binding PNP inhibitors show exceptionally long lifetimes on the target enzyme. We solve the mechanism of the target residence time by comparing functional off-rates in vitro and in vivo. We report in vitro PNPinhibitor dissociation rates (t(1/2)) from 3 to 31 min for seven Immucillins with dissociation constants of 115 to 6 pM. Treatment of human erythrocytes with DADMe-Immucillin-H (DADMe-ImmH, 22 pM) causes complete inhibition of PNP. Loss of [C-14] DADMe-ImmH from erythrocytes during multiple washes is slow and biphasic, resulting from inhibitor release and rebinding toPNPcatalytic sites. The slow phase gave a t(1/2) of 84 h. Loss of [C-14] DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t(1/2) of 1.6 h by preventing rebinding. Thus, in human erythrocytes, rebinding of DADMe-ImmH is 50-fold more likely than diffusional loss of the inhibitor from the erythrocyte. Humans treated with a single oral dose of DADMe-ImmH in phase 1 clinical trials exhibit regain of PNP activity with a t(1/2) of 59 days, corresponding to the erythropoiesis rate in humans. Thus, the PNP catalytic site recapture of DADMe-ImmH is highly favored in vivo. We conclude that transition-state analogues with picomolar dissociation constants exhibit long lifetimes on their targets in vivo because the probability of the target enzyme recapturing inhibitor molecules is greater than diffusional loss to the extracellular space.