Discovery and Validation of a Novel Class of Small Molecule Inhibitors of the CDC7 Kinase: Modulation of Tumor Cell Growth in Vitro and In Vivo.
Discovery and Validation of a Novel Class of Small Molecule Inhibitors of the CDC7 Kinase: Modulation of Tumor Cell Growth in Vitro and In Vivo.
复制标题
一类新型 CDC7 激酶小分子抑制剂的发现和验证：体外和体内肿瘤细胞生长的调节。
DOI：
10.1182/blood.v114.22.3771.3771
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
T. Kelly
中科院分区：
文献类型：
--
作者：
M. Frattini;David Shum;K. O'Dwyer;R. Brentjens;P. Maslak;M. Heaney;J. Jurcic;Hakim Djaballah;T. Kelly
摘要3771
海报板III-707
高通量筛选组成纪念斯隆-凯特琳化学库的化合物导致了几个确认的命中重组的CDC7：DBF4异二聚体激酶，一个在启动DNA复制和G1到S相变的关键调节因子。对HITS的化学信息学分析显示，由几种天然化合物组成的一个化学簇中存在丰富的物质，其中最有效的化合物CKI-7被选中进行进一步研究。首先，CKI-7被发现是ATP的非竞争性抑制剂，并促使我们将其与一组200个已知的激酶进行比较，以评估其选择性。结果与预期不谋而合，很少有激酶受到特异性影响。其次，CKI-7细胞毒活性针对一组既代表血液系统恶性肿瘤又代表实体肿瘤的成熟癌细胞系以及一组来自白血病患者的原代造血细胞(化疗初期和复发/难治性样本)进行了评估，结果发现CKI-7是一种非常有效的药物，其潜力在低纳摩尔范围内。随后利用一对高表达BclxL抗凋亡蛋白的同基因细胞株的研究进一步证实，在没有BclxL存在的情况下，通过激活caspase-3来诱导固有的细胞凋亡途径，而在BclxL存在的情况下，Caspase-3的活性减弱。标准的细胞周期同步化研究进一步证明了这一点，研究表明，接触CDC7抑制剂会导致S期停滞，细胞周期依赖的caspase-3激活，以及细胞凋亡。这种细胞死亡是CKI-7抑制CDC7激酶的直接结果，这一结果使用了CDC7底物生物标记物分析。第三，这类天然化合物的物理化学性质也促使我们研究它们对几种过表达多药耐药(MDR)细胞系的影响。我们发现CKI-7不是外排泵的底物，这表明这种新化合物可以克服人类肿瘤细胞对化疗耐药的一个主要机制。基于上述观察，CKI-7的体内抗肿瘤活性随后在使用最近分离的费城染色体阳性急性淋巴细胞白血病细胞系(PhALL3.1)的SCID-Beige小鼠系统性肿瘤模型中得到证实。综上所述，我们的数据证实了CDC7是一个新的有希望的癌症治疗靶点，而新发现的抑制剂CKI-7是一种天然存在的选择性小分子该酶抑制剂，也是一种同样有前景的新型癌症治疗剂。
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Abstract 3771
Poster Board III-707
High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naive and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl\_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl\_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.
Disclosures: No relevant conflicts of interest to declare.