Purine Pathways and Inhibitor Design in Plasmodium

描述(由申请人提供)：三十多年来，恶性疟原虫一直被认为是一种嘌呤营养缺乏症。尽管它与人类宿主不同，但我们对其嘌呤挽救途径和抗代谢产物的设计的了解已被证明不足以用于新的抗疟疾药物。嘌呤挽救途径将作为C.GRubmeyer(磷酸核糖基转移酶和抑制剂设计)、K.Kim(遗传学和表达)和V.L.Schramm(代谢和抑制剂设计)实验室之间的综合互动研究项目赠款(IRPG)的目标。IRPG的总体目标是：1)量化恶性疟原虫中嘌呤挽救的途径，2)表征基因敲除以确定主要挽救途径中特定步骤的消融路径反应，3)建立关键酶步骤的过渡状态，4)针对靶标设计和合成唯一有效的过渡状态抑制物，5)测试在人红细胞中培养的恶性疟原虫中的抑制物，以及6)比较在含有特定抑制物的培养物中的挽救途径与相同部位的基因敲除。这些结果将提供关于恶性疟原虫嘌呤途径的明确知识，必要步骤的基因测试，以及针对该途径中易受攻击的酶的强大的过渡态抑制剂。 随着~3H和~(14)C标记的前体进入核酸，嘌呤挽救途径将在培养的恶性疟原虫中被定量。高灵敏度加速器质谱仪(&gt；106比计数更灵敏)将被用来在不干扰嘌呤代谢物浓度的情况下确定嘌呤残留量。嘌呤回收通量将在嘌呤核苷磷酸化酶(PNP)、HGXPRT、APRT和甲硫腺苷磷酸化酶(MTAP)的基因破坏中进行比较。假说是PNP和HGXPRT形成了主要的嘌呤挽救途径。这条通路的消融可能导致腺苷挽救的第二条通路。过渡态抑制剂将在本实验室设计、合成和测试用于PNP和MTAP，以及用于APRT和HGPRT(格鲁梅耶实验室)。将测试过渡状态抑制剂对恶性疟原虫在红细胞中生长的影响，包括补充和不补充嘌呤以绕过代谢障碍。途径阻断的效率将通过比较基因敲除中的代谢物掺入模式和使用过渡态抑制剂的代谢物掺入模式来评估。对嘌呤回收途径中基本步骤的验证将指导本项目和未来的抑制剂设计。在嘌呤回收的关键步骤诱导抑制剂阻断可能为杀灭疟疾寄生虫提供一种有效的策略。

DESCRIPTION (provided by the applicant): Plasmodium falciparum has been recognized as a purine auxotroph for more than three decades. Despite this difference from its human host, our understanding of its purine salvage pathways and the design of anti-metabolites have proven inadequate for new antimalarial agents. The purine salvage pathways will be targeted in an integrated Interactive Research Project Grant (IRPG) between the laboratories of C. Grubmeyer (phosphoribosyltransferases and inhibitor design), K. Kim (genetics and expression) and V.L. Schramm (metabolism and inhibitor design). The overall goals of the IRPG are to: 1) quantitate the pathways of purine salvage in Plasmodium falciparum, 2) characterize genetic knock-outs to define the pathway response to ablation of specific steps in major salvage pathways, 3) establish the transition states of the critical enzymatic steps, 4) design and synthesize uniquely powerful transition state inhibitors against the targets, 5) test the inhibitors in P. falciparum cultured in human erythrocytes, and 6) compare the salvage pathways in cultures with specific inhibiors to gene knock-outs at the same sites. The results will provide definitive knowledge of the purine pathways of P. falciparum, genetic tests of essential steps and powerful transition state inhibitors against vulnerable enzymes of the pathway. Purine salvage pathways will be quantitated in cultured P. falciparum by following 3H and 14C-labeled precursors into nucleic acids. High-sensitivity accelerator mass spectrometry (>106 more sensitive than counting) will be used to establish purine salvage flux without perturbing purine metabolite concentrations. Purine salvage flux will be compared in gene-disruptions of purine nucleoside phosphorylase (PNP), HGXPRT, APRT, and methylthioadenosine phosphorylase (MTAP). The hypothesis is that PNP and HGXPRT form the dominant purine salvage pathway. Ablation of this pathway may induce a secondary pathway of adenosine salvage. Transition state inhibitors will be designed, synthesized and tested for PNP and MTAP in this lab, and for APRT and HGPRT (Grubmeyer lab). The effects of transition state inhibitors will be tested on the growth of P. falciparum in erythrocytes with and without purine supplements to by-pass the metabolic block. The efficiency of pathway blocks will be evaluated by comparing metabolite incorporation patterns in genetic knockouts to those with transition state inhibitors. Validation of essential steps in purine salvage pathways will direct this and future programs in inhibitor design. Inducing inhibitor blocks at essential steps of purine salvage may provide an effective strategy for killing the malarial parasite.