Cody Vivian, Luft Joseph R, Pangborn Walter, Gangjee Aleem
Structural Biology Department, Hauptman-Woodward Medical Research Institute Inc., 73 High Street, Buffalo, NY 14203, USA.
Acta Crystallogr D Biol Crystallogr. 2003 Sep;59(Pt 9):1603-9. doi: 10.1107/s0907444903014963. Epub 2003 Aug 19.
Structural data are reported for the first example of the potent antifolate inhibitor 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxy-N-methylanilino)methyl]pyrido[2,3-d]pyrimidine (1) in complex with human dihydrofolate reductase (hDHFR) and NADPH. Small differences in crystallization conditions resulted in the growth of two different forms of a binary complex. The structure determination of an additional crystal of a ternary complex of hDHFR with NADPH and (1) grown under similar conditions is also reported. Diffraction data were collected to 2.1 A resolution for an R3 lattice from a hDHFR ternary complex with NADPH and (1) and to 2.2 A resolution from a binary complex. Data were also collected to 2.1 A resolution from a binary complex with hDHFR and (1) in the first example of a tetragonal P4(3)2(1)2 lattice. Comparison of the intermolecular contacts among these structures reveals differences in the backbone conformation (1.9-3.2 A) for flexible loop regions (residues 40-46, 77-83 and 103-107) that reflect differences in the packing environment between the rhombohedral and tetragonal space groups. Analysis of the packing environments shows that the tetragonal lattice is more tightly packed, as reflected in its smaller V(M) value and lower solvent content. The conformation of the inhibitor (1) is similar in all structures and is also similar to that observed for TMQ, the parent quinazoline compound. The activity profile for this series of 5-deaza N-substituted non-classical trimethoxybenzyl antifolates shows that the N10-CH(3) substituted (1) has the greatest potency and selectivity for Toxoplasma gondii DHFR (tgDHFR) compared with its N-H or N-CHO analogs. Models of the tgDHFR active site indicate preferential contacts with (1) that are not present in either the human or Pneumocystis carinii DHFR structures. Differences in the acidic residue (Glu30 versus Asp for tgDHFR) affect the precise positioning of the diaminopyridopyrimidine ring, while changes in other residues, particularly at positions 60 and 64 (Leu versus Met and Asn versus Phe), involve interactions with the trimethoxybenzyl substituents.
报道了强效抗叶酸抑制剂2,4 - 二氨基 - 5 - 甲基 - 6 - [(3',4',5'-三甲氧基 - N - 甲基苯胺基)甲基]吡啶并[2,3 - d]嘧啶（1）与人类二氢叶酸还原酶（hDHFR）和NADPH形成复合物的首个结构数据。结晶条件的微小差异导致了两种不同形式的二元复合物的生长。还报道了在相似条件下生长的hDHFR与NADPH和（1）的三元复合物的另一个晶体的结构测定。从hDHFR与NADPH和（1）的三元复合物收集了分辨率为2.1 Å的衍射数据用于R3晶格，从二元复合物收集的数据分辨率为2.2 Å。在四方P4(3)2(1)2晶格的首个例子中，也从与hDHFR和（1）的二元复合物收集了分辨率为2.1 Å的数据。这些结构之间分子间接触的比较揭示了柔性环区域（残基40 - 46、77 - 83和103 - 107）主链构象（1.9 - 3.2 Å）的差异，这反映了菱面体和四方空间群之间堆积环境的差异。堆积环境分析表明四方晶格堆积更紧密，这体现在其较小的V(M)值和较低的溶剂含量上。抑制剂（1）在所有结构中的构象相似，也与母体喹唑啉化合物TMQ观察到的构象相似。这一系列5 - 脱氮N - 取代非经典三甲氧基苄基抗叶酸药物的活性谱表明，与N - H或N - CHO类似物相比，N10 - CH(3)取代的（1）对刚地弓形虫二氢叶酸还原酶（tgDHFR）具有最大的效力和选择性。tgDHFR活性位点模型表明与（1）存在优先接触，而在人类或卡氏肺孢子虫二氢叶酸还原酶结构中不存在这种接触。酸性残基的差异（tgDHFR为Glu30而不是Asp）影响二氨基吡啶并嘧啶环的精确定位，而其他残基的变化，特别是在60和64位（Leu对Met以及Asn对Phe），涉及与三甲氧基苄基取代基的相互作用。
