Enzyme inhibition, polyglutamation, and the effect of LY231514 (MTA) on purine biosynthesis.
Enzyme inhibition, polyglutamation, and the effect of LY231514 (MTA) on purine biosynthesis.
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发表时间：
1999-04
影响因子：
4
通讯作者：
L. G. Mendelsohn;Chuan Shih;V. Chen;L. L. Habeck-L.;S. B. Gates;K. Shackelford
中科院分区：
文献类型：
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作者：
L. G. Mendelsohn;Chuan Shih;V. Chen;L. L. Habeck-L.;S. B. Gates;K. Shackelford
基于吡咯并嘧啶的抗叶酸剂，N-4-[2-（2-氨基-3，4-二氢-4-氧代-7H-吡咯并[2，3-d]嘧啶-5-基）乙基]苯甲酰基谷氨酸，LY 231514（MTA）已在广泛的人类肿瘤中表现出抗肿瘤活性，包括乳腺癌、结肠癌、非小细胞肺癌、头颈癌、胰腺癌和其他实体瘤。这种活性的生化基础进行了探讨，通过测量激活MTA的多聚谷氨酸和活性的MTA抑制几个叶酸依赖性酶：胸苷酸合成酶，二氢叶酸还原酶，甘氨酰胺核糖核苷酸甲酰转移酶（GARFT）。叶酸聚谷氨酸合酶（FPGS）非常有效地激活MTA。与另一种底物洛美沙星相比，用两种不同物种的FPGS，得到的Km值低于2 μ mol/L和较高的相对一级速率常数k'（Vmax/Km）分别为6.4和13.7。在体外比较了几种叶酸拮抗剂在高和低底物浓度下聚谷氨酸的形成。在低MTA浓度下，四谷氨酸和五谷氨酸MTA是24小时孵育期后鉴定的主要形式。相反，在相同条件下，仅形成二谷氨酰甲氨蝶呤和GARFT抑制剂洛美沙星的三谷氨酰化、四谷氨酰化和五谷氨酰化形式的混合物。在较高的底物浓度（20 μ mol/L，24小时），更大量的每种产品形成。然而，主要代谢产物为三谷氨酸MTA或三谷氨酸洛美沙星，而仅回收了二谷氨酰甲氨蝶呤。因此，MTA是FPGS的优良底物，并且通过该酶将其有效代谢为高度聚谷氨酸化的物质。测定MTA及其聚谷氨酸化代谢物抑制几种叶酸依赖性酶的活性。在体外，MTA和它的聚谷氨酸盐是几种叶酸依赖性酶的有效的紧密结合抑制剂，包括胸苷酸合成酶、二氢叶酸还原酶和GARFT。初步的基于细胞的测定（CCRF-CEM）证明MTA对嘌呤从头途径的抑制作用，与其对肿瘤细胞的多靶向作用机制一致。MTA活化为高度聚谷氨酸化代谢物的联合作用和这些聚谷氨酸抑制多种叶酸依赖性酶的效力为理解该化合物对许多人类肿瘤类型的广泛抗肿瘤活性提供了机制基础。
The pyrrolopyrimidine-based antifolate, N-¿4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]benzoyl¿glutamic acid, LY231514 (MTA) has demonstrated antitumor activity in a broad array of human tumors, including breast cancer, colon cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, and other solid tumors. The biochemical basis of this activity was explored by measuring activation of MTA by polyglutamation and the activity of MTA to inhibit several folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). The enzyme folylpolyglutamate synthase (FPGS) activated MTA very efficiently. Using FPGS from two different species, Km values below 2 micromol/L and high relative first order rate constants, k' (Vmax/Km) of 6.4 and 13.7 compared with another substrate, lometrexol, were obtained. The formation of polyglutamates of several antifolates were compared in vitro at high and low substrate concentrations. At low MTA concentrations, tetraglutamated and pentaglutamated MTA were the predominant forms identified after a 24-hour incubation period. In contrast, only diglutamyl methotrexate and a mixture triglutamylated, tetraglutamylated, and pentaglutamylated forms of the GARFT inhibitor lometrexol were formed under the same conditions. At higher substrate concentrations (20 micromol/L, 24 hours), greater amounts of each product were formed. The major metabolites, however, were triglutamated MTA or triglutamated lometrexol, while only diglutamyl methotrexate was recovered. Thus, MTA was an excellent substrate for FPGS and it was efficiently metabolized to highly polyglutamated species by this enzyme. The activity of MTA and its polyglutamated metabolites to inhibit several folate-dependent enzymes was measured. In vitro, MTA and its polyglutamates were potent, tight-binding inhibitors of several folate-dependent enzymes, including thymidylate synthase, dihydrofolate reductase, and GARFT. Preliminary cell-based assays (CCRF-CEM) demonstrated inhibition of the purine de novo pathway by MTA, consistent with its multitargeted mechanism of action against tumor cells. The combined effects of activation of MTA to highly polyglutamated metabolites and the potency of these polyglutamates to inhibit multiple folate-dependent enzymes provide a mechanistic basis for understanding the broad antitumor activity of this compound against many human tumor types.