In vitro evaluation of a novel 2,6,9-trisubstituted purine acting as a cyclin-dependent kinase inhibitor
In vitro evaluation of a novel 2,6,9-trisubstituted purine acting as a cyclin-dependent kinase inhibitor
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DOI：
10.1111/j.1749-6632.1999.tb09411.x
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发表时间：
1999-01-01
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影响因子：
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通讯作者：
Favaudon, V
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文献类型：
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作者：
Giocanti, N;Sadri, R;Favaudon, V
癌症中细胞周期进程的频繁失调1促使人们积极寻找对细胞周期蛋白依赖性激酶（Cdks）具有高亲和力和特异性的激酶抑制剂。到目前为止，已经确定了三大类cdk靶向药物，包括丁内酯I， 2多羟基黄酮，如黄哌啶醇，3和取代嘌呤。4第一个取代嘌呤衍生物作为选择性Cdk抑制剂，olomoucine，已经通过筛选Cdk1/cyclin B复合物被确定。Olomoucine竞争性地抑制Cdk1， Cdk2, Cdk5，并在较小程度上抑制Erk1。最近的研究结果表明，2,6,9 -三取代嘌呤的药理学性质出乎意料，这些嘌呤是由奥莫乌辛铅结构衍生的。为了更详细地研究这个问题，我们开发了一个程序来合成和评价这个系列中的新化合物。从海星卵母细胞和人重组Cdk5/p35复合物中合成27个衍生物，并测定其对Cdk1/cyclin B的特异性抑制作用。与先前的结果一致，5数据显示对Cdk1和Cdk5的抑制效率之间存在很强的相关性。相比之下，所有化合物对Erk1和Erk2激酶仅具有轻微活性。该系列中的一种化合物ML-1437对纯化的Cdk1/cyclin B、Cdk5/p35和Cdk2/cyclin e的活性比olomoucine高得多，它在体外对人宫颈癌HeLa细胞也显示出明显的细胞毒性，即使是短时间暴露。生长的IMR-90（人正常成纤维细胞）、LoVo（人结肠腺癌）和SQ-20B（人头颈部鳞状癌）细胞的结果相似，但随着细胞（SQ-20B和IMR-90）达到融合，耐药性迅速增加。这些结果表明，对Cdks的亲和力和药物的细胞毒性潜力是相互关联的（图1，表1）。除了同步HeLa细胞S期早期S期过渡明显延长外，亚毒浓度的ML-1437被证明不能对细胞周期进程产生可逆的阻滞。观察发现，细胞周期的G1和G2期阻滞与诱导细胞死亡相关，长期暴露于致死剂量的药物导致与有丝分裂细胞死亡相关的大量微核，没有证据表明存在内复制（多倍体化）或ap-
The frequent deregulation of cell cycle progression in cancer1 has prompted an active search for kinase inhibitors with high affinity and specificity for cyclin-dependent kinases (Cdks). Three major classes of Cdk-targeting drugs have been identified to date, including butyrolactone I, 2 polyhydroxylated flavones such as flavopiridol, 3 and substituted purines. 4 The first substituted purine derivative acting as a selective Cdk inhibitor, olomoucine, has been identified from screening against Cdk1/cyclin B complex. 5 Olomoucine competitively inhibits Cdk1, Cdk2, Cdk5, and, to a lesser extent, Erk1. 5Recent results have pointed to unexpected pharmacologic properties of 2, 6, 9-trisubstituted purines derived from the olomoucine lead structure. 6, 7 To investigate the question in more detail, we developed a program for synthesis and evaluation of new compounds in this series. Twenty-seven derivatives were synthesized and assayed for specific inhibition of Cdk1/cyclin B from starfish oocytes and human recombinant Cdk5/p35 complex. In agreement with earlier results, 5 data showed that a strong correlation exists between inhibitory efficiencies against Cdk1 and Cdk5. In contrast, all compounds were only marginally active against Erk1 and Erk2 kinases. One compound in the series, ML-1437, proved much more active than olomoucine against purified Cdk1/cyclin B, Cdk5/p35, and Cdk2/cyclin E. It also showed pronounced cytotoxicity against human cervix carcinoma HeLa cells in vitro, even on short exposure. Growing IMR-90 (human normal fibroblasts), LoVo (human colon adenocarcinoma), and SQ-20B (human head and neck squamous carcinoma) cells gave similar results, but drug resistance increased rapidly as cells (SQ-20B and IMR-90) reached confluence. These results suggest that the affinity for Cdks and the cytotoxic potential of the drugs are interrelated (FIG. 1, TABLE 1). With the exception of pronounced lengthening of S phase transit during early-S in synchronized HeLa cells, ML-1437 at subtoxic concentration proved unable to produce reversible arrest of the cell cycle progression. When observed, arrest in the G1 and G2 phases of the cell cycle correlated with induced cell death, and chronic exposure to lethal doses of the drug resulted in massive micronucleation in relation to mitotic cell death, with no evidence of endoreduplication (polyploidization) or ap-