Purine Pathways and Inhibitor Design in Plasmodium

描述（由申请人提供）：恶性疟原虫已被认为是一种嘌呤营养不良超过三十年。尽管与人类宿主存在这种差异，但我们对其嘌呤回收途径和抗代谢物设计的理解已被证明不足以开发新的抗疟药。嘌呤回收途径将在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）实验室的综合互动研究项目资助（IRPG）中进行。小组的整体目标是：1)量化恶性疟原虫嘌呤回收途径；2)表征基因敲除，以确定主要回收途径中特定步骤消失时的途径反应；3)建立关键酶步骤的过渡状态；4)设计和合成针对靶点的独特强大的过渡状态抑制剂；5)在人红细胞培养的恶性疟原虫中测试抑制剂。6)比较具有特定抑制剂的培养液对相同位点基因敲除的挽救途径。该结果将提供恶性疟原虫嘌呤途径的明确知识，关键步骤的基因测试和针对该途径易感酶的强大过渡状态抑制剂。

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.