STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

二氢叶酸还原酶（DHFR）是“抗叶酸”药物的靶标， 在临床上可用于治疗癌症、疟疾和细菌感染， 感染. 这种治疗原生动物和细菌的功效 由于出现了耐药微生物， 具有变体DHFR，对于至少一些变体，解离常数 与野生型DHFR相比，抑制剂的K/d大大增加 (wt)而二氢叶酸（H2 folate）的K/m和k（cat）则小得多 影响。 类似的DHFR抗性形式也已从 在培养物中暴露于抗叶酸剂的哺乳动物细胞。 所有已知的突变 对来自任何物种的DHFR赋予抗性发生在八个位点之一 在蛋白质序列中，但只有少数突变之一的影响， 在大多数这些地点都被探索过。 在这个项目中，我们将探索 在这些位点的其他突变的影响。 具体来说，我们将在 人DHFR（hDHFR）突变对甲氨蝶呤敏感性的影响 （MTX）。 盒替换方法将用于引入突变 我们已经使用了一个高表达载体， 重组hDHFR的表达。 对MTX高度耐药的突变hDHFR将被广泛应用于临床。 其特征在于关于结合的动力学和热力学， 底物，产物和MTX，催化动力学，和动力学， 蛋白质稳定性的热力学 合作安排已 用于这些突变体复合物的X射线晶体学。 我们还将 研究MTX以外的任何抑制剂是否与 这些突变hDHFR的修饰的活性位点。 在该项目的另一部分，我们将检查白血病细胞中的hDHFR 从接受标准治疗后复发的儿科患者中， 用大剂量MTX反复治疗。 从mRNA逆转录， 随后从单链cDNA进行PCR，将用于制备cDNA 并将其克隆到高表达载体中。 hDHFR 将检查获得的样品的MTX抑制作用。 如果具有高Ki的酶是 发现负责的突变将通过以前的方法进行研究 指出了

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.