STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

二氢叶酸还原酶(DHFR)是抗叶酸药物的靶标 在治疗癌症、疟疾和细菌方面临床上很有用 感染。这样治疗原虫和细菌的疗效观察 由于出现了耐药生物，感染情况有所减少 具有不同的DHFRs，其中至少一部分的解离常数 (K/d)与野生型DHFR相比有很大提高 (WT)，而二氢叶酸(H_2叶酸)的K/m和k(CAT)要小得多 受影响。类似的耐药形式的dhfr也从 哺乳动物细胞在培养中暴露于反叶酸。所有已知的突变 任何物种对DHFR的抗性都发生在八个位点中的一个 在蛋白质序列中，但只有少数几个突变中的一个的影响 在这些遗址中的大多数都被探索过。在本项目中，我们将探索 这些位点的其他突变的影响。具体来说，我们将在 人DHFR(HDHFR)基因突变对甲氨蝶呤敏感性的影响 (MTX)。盒式磁带更换方法将被用来引入突变 在我们已经使用的高效表达载体中转化为hDHFR基因 Wt hDHFR的表达。 对MTX高度耐药的突变型hDHFRs将被广泛 关于结合的动力学和热力学的特征的 底物，产物和甲氨蝶呤，催化动力学，动力学和 蛋白质稳定性的热力学。合作安排一直是 用于这些突变体的复合体的X射线结晶学研究。我们还将 调查除MTX外，是否有其他抑制剂与 这些突变型hDHFRs的修饰活性部位。 在项目的另一部分中，我们将检测白血病细胞中的hDHFR。 来自复发的标准治疗的儿科患者，包括 重复大剂量甲氨蝶呤治疗。从信使核糖核酸反转录， 然后从单链cDNA中进行聚合酶链式反应，将其用于制备cDNA 为hDHFR，将其克隆入高效表达载体。Hdhfr 获得的药物将由甲氨蝶呤进行抑制试验。如果具有高KI的酶 发现了导致突变的原因将通过前面的方法进行研究 已注明。

Dihydrofolate reductase (DHFR) is the target for "antifolate" drugs that are clinically useful in treatment of cancer, malaria, and bacterial infections. Efficacy of such treatment of protozoal and bacterial infections has been decreased by the appearance of resistant organisms having variant DHFRs, for at least some of which the dissociation constant (K/d) for the inhibitor is greatly increased compared with wild-type DHFR (wt) while K/m for dihydrofolate (H2folate), and k(cat) are much less affected. Similar resistant forms of DHFR have also been purified from mammalian cells exposed to antifolates in culture. All known mutations conferring resistance on DHFR from any species occur at one of eight sites in the protein sequence, but the effect of only one of a few mutations has been explored at most of these sites. In this project we will explore the effect of other mutations at these sites. Specifically, we will examine in human DHFR (hDHFR) affects of such mutations on sensitivity to methotrexate (MTX). The cassette replacement method will be used to introduce mutations into hDHFR cDNA in a high expression vector that we have already used for the expression of wt hDHFR. Mutant hDHFRs that are highly resistant to MTX will be extensively characterized with respect to kinetics and thermodynamics of the binding of substrates, products and MTX, the kinetics of catalysis, and kinetics and thermodynamics of protein stability. Collaborative arrangements have been made for X-ray crystallography of complexes of these mutants. We will also investigate whether any inhibitors other than MTX bind tightly to the modified active site of these mutant hDHFRs. In another part of the project, we will examine hDHFR from leukemic cells from pediatric patients who have relapsed on standard therapy that includes repeated treatment with high dose MTX. Reverse transcription from mRNA, followed by PCR from the single strand cDNA, will be used to prepare cDNA for hDHFR and this will be cloned into a high expression vector. the hDHFR obtained will be examined for inhibition by MTX. If enzyme with high Ki is found the mutation responsible will be studied by the methods previously indicated.