Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; Effect of Tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells
Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; Effect of Tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells
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DOI：
10.1016/0006-2952(96)00035-4
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发表时间：
1996-05-17
影响因子：
5.8
通讯作者：
Jackman, AL
中科院分区：
文献类型：
--
作者：
Aherne, GW;Hardcastle, A;Jackman, AL
近年来，抑制胸苷酸合成酶（TS）作为药物开发的靶点备受关注，已有多个化合物进入临床评价阶段。在药物开发期间，可通过测定药物处理细胞中脱氧胸苷5-三磷酸（TTP）和脱氧尿苷5 '-单磷酸（dUMP）池的扰动来评估靶点抑制的有效性。快速，灵敏，和重复性的TTP池和免疫反应性dUMP池的放射免疫测定已经开发，以满足我们的要求，快速评估TS抑制喹唑啉抗叶酸剂。该分析可以在1-2百万个细胞上进行，并且需要最少的样品制备。TTP和免疫反应性dUMP的检测限分别为1 pmole/10（6）个细胞和1 pmole/10（6）个细胞。在用喹唑啉抗叶酸剂ZD 1694处理的小鼠L1210白血病细胞中测量了TTP和“dUMP "池（N-（5-[N-（3，4-二氢-2-甲基-4-氧代喹唑啉-6-基甲基）-N-甲胺]-2-噻吩甲酰基）-L-谷氨酸）和CB 30900（N-[N-[4-[N-[3，4-二氢-2，7-二甲基-4-氧代-6-喹唑啉基）甲基]-N-丙-2-炔基氨基]-2-氟苯甲酰基]-L-γ谷氨酰基]-D-谷氨酸）。与ZD 1694不同，CB 30900是一种TS抑制剂，其活性不依赖于聚谷氨酸化。在L1210细胞中，这两种化合物以剂量和时间相关的方式引起TTP池的快速抑制。在等毒性剂量下暴露于每种化合物4小时后，实现了大于90%的TS抑制（高达100倍的IC 50，通过48小时生长抑制试验测定）。对于两种化合物，这伴随着“dUMP "池增加5-10倍。对于ZD 1694，当细胞在无药物培养基中重悬4小时时，TTP合并液或“dUMP "水平均未正常化，并且在研究的较高剂量下，在不存在药物的情况下16小时后，TS仍受到抑制。这与2D 1694的有效多谷氨酸化形式的形成和细胞内保留一致。相比之下，通过TTP池的补充和“dUMP "水平的标准化确定的CB 30900的TS活性得到证实。然而，在高剂量（50 μ M，相当于250倍的IC 50），TS抑制的保留后4小时，但不是16小时，在没有药物的情况下观察到。所描述的放射免疫测定将证明可用于进一步确定新型抗叶酸剂化合物对TS抑制的程度和时间过程，并且还将提供关于单独使用或与其他药物和调节剂组合使用时已建立的抗代谢物的有价值的体外和体内药效学信息。
The inhibition of thymidylate synthase (TS) as a drug development target has received much attention in recent years, and several compounds have reached clinical evaluation. During drug development, the effectiveness of target inhibition can be assessed by determination of the perturbations of deoxythymidine 5-triphosphate (TTP) and deoxyuridine 5'-monophosphate (dUMP) pools in drug-treated cells. Rapid, sensitive, and reproducible radioimmunoassays for TTP pools and immunoreactive dUMP pools have been developed to meet our requirement for the rapid assessment of TS inhibition by quinazoline antifolates. The assays can be carried out on 1-2 million cells, and require minimal sample preparation. The limit of detection for TTP is 1 pmole/10(6) cells and for immunoreactive dUMP (''dUMP''), 3.0 pmole/10(6) cells, both assays being performed on the same cell extract.TTP and ''dUMP'' pools have been measured in mouse L1210 leukaemia cells treated with the quinazoline antifolates ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamine]-2-thenoyl)-L-glutamic acid) and CB30900 (N-[N-[4-[N-[3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-fluorobenzoyl]-L-gamma glutamyl]-D-glutamic acid). Unlike ZD1694, CB30900 is a TS inhibitor that does not rely on polyglutamation for activity. In L1210 cells, both compounds caused a rapid inhibition of TTP pools in a dose- and time-related manner. Greater than 90% TS inhibition was achieved following a 4-hr exposure to each compound at equitoxic doses (up to 100 times the IC50 determined by a 48-hr growth inhibition assay). For both compounds, this was accompanied by a 5-10 fold increase in ''dUMP'' pools. For ZD1694, neither the TTP pool or ''dUMP'' levels were normalised when cells were resuspended in a drug-free medium for 4 hr and, at the higher doses studied, TS was still inhibited after a 16-hr period in the absence of drug. This is consistent with the formation and intracellular retention of potent polyglutamated forms of 2D1694. In contrast, TS activity as determined by repletion of the TTP pools and normalisation of ''dUMP'' levels were demonstrated for CB30900. However, at a high dose (50 mu M, equivalent to 250 times the IC50), retention of TS inhibition was observed following 4 hr, but not 16 hr in the absence of drug. The radioimmunoassays described will prove useful to further define the extent and time-course of TS inhibition by novel antifolate compounds, and will also provide valuable in vitro and in vivo pharmacodynamic information on established antimetabolites when used alone or in combination with other drugs and modulators.