STRUCTURAL STUDIES OF AIDS RESPONSIVE DRUGS

描述（由申请人提供）：卡氏肺孢子虫（pc）和弓形虫 弓形虫（tg）是机会性感染和死亡的主要原因， 免疫抑制患者，特别是艾滋病患者。抗叶酸药物， 通常由磺酰胺与甲氧苄啶组合组成， 二氢叶酸还原酶（DHFR）的抑制剂，一直是最 目前临床使用的有效药物。然而，它们的使用受到限制， 毒性和抗性的问题。该项目的主要目标是 二氢叶酸三维晶体结构的测定 来自真菌（卡氏肺孢子虫，pc）、原生动物（弓形虫）的DHFR 弓形虫、TG）和哺乳动物（大鼠肝、小鼠和人）来源作为复合物 用对PC或TG酶显示选择性和特异性的抗叶酸剂。 其目的是比较抗叶酸剂-酶相互作用的结构细节， 为了设计更有选择性和更有效的药物， 机会性感染导致肺炎，这是死亡的主要原因， 艾滋病患者。作为该协议的一部分，我们计划利用这些结构 新的选择性抗叶酸剂的设计和合成的数据。六项具体 目的是为了检验这一假设，即抗叶酸剂的使用， 防治卡氏肺孢子虫或弓形虫的机会性感染 弓形虫生物体是特定酶-抑制剂相互作用的结果。我们将 分析：（1）第一个人源性pcDHFR抑制剂复合物，以检查 对配体结合的影响是由显著的序列变化引起的 种属间差异，（2）首次验证大鼠肝脏DHFR复合物 抑制与人DHFR的相关性，（3）第一个tgDHFR复合物，（4） 小鼠DHFR，用于与人DHFR比较，（5）DHFR与新的 抗叶酸剂，和（6）同源性建模数据，以了解控制 抗叶酸选择性。将利用这些研究所获得的知识 设计和合成新的抗叶酸剂。适当的目标是 选择用于各种DHFR酶的研究。对这些数据的分析将 提供了分子水平上的细节，如底物-酶的几何结构，氢键， 构象和特定活性位点残基的作用，特别是 通过在人和人之间的位置31和64处的取代的贡献 pcDHFR在调节pc选择性中的作用。因为选择性显然只需要 酶抑制剂几何形状的微小变化，我们建议寻找微妙的 一系列仔细测定的DHFR晶体结构的差异 与抗叶酸剂的复合物对特定物种显示出选择性 DHFR。因此，了解酶抑制剂的三维结构 需要复合物来确定DHFR选择性和作用的机制。 印第安纳州大学的Sherry Queener博士将测量 选择抗叶酸剂。

DESCRIPTION (provided by applicant): Pneumocystis carinii (pc) and toxoplasma gondii (tg) are major causes of opportunistic infection and mortality in immuno-suppressed patients, particularly those with AIDS. Antifolate drugs, usually consisting of a sulfonamide in combination with trimethoprim, an inhibitor of the enzyme dihydrofolate reductase (DHFR), have been the most effective drugs in clinical use to date. However, their use has been limited by problems of toxicity and resistance. The major goal of this project is determination of the three-dimensional crystal structures of dihydrofolate reductase (DHFR) from fungal (Pneuntocystis carinii, pc), protozoal (toxoplasma gondii, tg), and mammalian (rat liver, mouse and human) sources as complexes with antifolates that show selectivity and specificity for the pc or tg enzyme. The aim is to compare structural details of antifolate-enzyme interactions in order to design more selective and potent agents as effective treatment for opportunistic infections that cause pneumonia, a major cause of mortality among AIDS patients. As part of this protocol we plan to exploit these structural data for the design and synthesis of new selective antifolates. Six specific aims are proposed to test the hypothesis that efficacy of antifolate use in combating opportunistic infections from Pneumocystis carinii or toxoplasma gondii organisms is a result of specific enzyme-inhibitor interactions. We will analyze: (1) the first human-derived pcDHFR inhibitor complexes to examine the effects on ligand binding that result from the significant sequence changes between species, (2) the first rat liver DHFR complexes to validate correlations of inhibition with human DHFR, (3) the first tgDHFR complexes, (4) mouse DHFR for comparison to human DHFR, (5) DHFR complexes with novel antifolates, and (6) homology modeling data to understand features that control antifolate selectivity. The knowledge gained by these studies will be utilized in the design and synthesis of new antifolates. Appropriate targets have been selected for study with various DHFR enzymes. Analysis of these data will provide molecular level details of inhibitor-enzyme geometry, hydrogen bonding, conformation and the role of specific active site residues, especially the contribution by the substitution at positions 31 and 64 between human and pcDHFR in modulating pc selectivity. Since selectivity apparently requires only small changes in enzyme-inhibitor geometry, we propose to look for subtle differences in a series of carefully determined crystal structures of DHFR complexes with antifolates that show selectivity to a particular species of DHFR. Thus knowledge of the three dimensional structure of enzyme-inhibitor complexes are required to define the mechanism of DHFR selectivity and action. Dr. Sherry Queener, Indiana University, will measure inhibitory activity of selected antifolates.