Thorndike J, Gaumont Y, Kisliuk R L, Sirotnak F M, Murthy B R, Nair M G, Piper J R
Department of Biochemistry, Tufts University Health Science Campus, Boston, Massachusetts 02111.
Cancer Res. 1989 Jan 1;49(1):158-63.
In order to determine the biochemical basis for the cytotoxicity of homofolates, poly-gamma-glutamyl derivatives of homofolate (HPteGlu) and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT), thymidylate synthase, and serine hydroxymethyltransferase (SHMT) in extracts of Manca human lymphoma and L1210 murine leukemia cells. The most striking inhibitions are that of GARFT by (6R,S)-H4HPteGlu4-6 with IC50 values from 1.3 to 0.3 microM. Both diastereomers, (6R)-H4HPteGlu6 and (6S)-H4HPteGlu6, inhibit GARFT activity. In Manca cell extracts, the (6S) form is more potent than the (6R) form whereas in the murine system the reverse is true. The (6R,S)-H4HPteGlu polyglutamates are weak inhibitors of human AICARFT (IC50, 6-10 microM). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGlu4-6 having IC50 values close to 2 microM. Polyglutamates of HPteGlu and of H4HPteGlu are weaker inhibitors of thymidylate synthase (IC50, 8 microM for HPteGlu5-6 and greater than 20 microM for H4HPteGlu1-5). Polyglutamates of HPteGlu and of H4HPteGlu are poor inhibitors of SHMT (IC50, greater than 20 microM). Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H4HPteGlu at 6 and 8 microM, respectively. Both of these effects are reversed by 0.1 mM inosine. Trimetrexate at a subinhibitory concentration, 10 nM, antagonizes growth inhibition by HPteGlu, raising the IC50 from 6 to 64 microM, but enhances inhibition by (6R,S)-5-methyl-H4HPteGlu, lowering the IC50 from 8 to 5 microM. Our results support the view that homofolates become toxic after conversion to H4HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis.
为了确定高叶酸盐细胞毒性的生化基础，合成了高叶酸盐（HPteGlu）和四氢高叶酸盐（H4HPteGlu）的聚γ-谷氨酰衍生物，并将其作为甘氨酰胺核糖核苷酸甲酰基转移酶（GARFT）、氨基咪唑甲酰胺核糖核苷酸甲酰基转移酶（AICARFT）、胸苷酸合成酶和丝氨酸羟甲基转移酶（SHMT）的抑制剂，在曼卡人淋巴瘤和L1210小鼠白血病细胞提取物中进行测试。最显著的抑制作用是(6R,S)-H4HPteGlu4-6对GARFT的抑制，IC50值为1.3至0.3微摩尔/升。两种非对映异构体，(6R)-H4HPteGlu6和(6S)-H4HPteGlu6，均抑制GARFT活性。在曼卡细胞提取物中，(6S)形式比(6R)形式更有效，而在小鼠系统中则相反。(6R,S)-H4HPteGlu多聚谷氨酸是人类AICARFT的弱抑制剂（IC50，6 - 10微摩尔/升）。然而，HPteGlu的多聚谷氨酸对AICARFT的抑制作用更强，HPteGlu4-6的IC50值接近2微摩尔/升。HPteGlu和H4HPteGlu的多聚谷氨酸是胸苷酸合成酶的较弱抑制剂（HPteGlu5-6的IC50为8微摩尔/升，H4HPteGlu1-5的IC50大于20微摩尔/升）。HPteGlu和H4HPteGlu的多聚谷氨酸是SHMT的较差抑制剂（IC50大于20微摩尔/升）。HPteGlu和(6R,S)-5-甲基-H4HPteGlu分别在6和8微摩尔/升时抑制曼卡细胞生长50%。这两种作用均可被0.1毫摩尔/升的肌苷逆转。亚抑制浓度10纳摩尔/升的三甲曲沙可拮抗HPteGlu对生长的抑制作用，将IC50从6微摩尔/升提高到64微摩尔/升，但增强(6R,S)-5-甲基-H4HPteGlu的抑制作用，将IC50从8微摩尔/升降低到5微摩尔/升。我们的结果支持这样一种观点，即高叶酸盐在转化为H4HPteGlu多聚谷氨酸后变得有毒，后者会阻断嘌呤生物合成中的一步——GARFT。
