Purine Pathways and Inhibitor Design in Plasmodium

描述（由申请方提供）：三十多年来，恶性疟原虫被认为是嘌呤营养缺陷型。尽管与人类宿主存在这种差异，但我们对其嘌呤补救途径的理解和抗代谢物的设计已被证明不足以用于新的抗疟疾药物。嘌呤补救途径将在一个综合的互动研究项目资助（IRPG）之间的实验室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）。IRPG的总体目标是：1）定量恶性疟原虫中嘌呤补救的途径，2）表征基因敲除以定义对主要补救途径中特定步骤的消除的途径响应，3）建立关键酶促步骤的过渡态，4）设计和合成针对靶标的独特强效过渡态抑制剂，5）测试在人红细胞中培养的恶性疟原虫中的抑制剂，以及6）比较在具有特异性抑制剂的培养物中的补救途径与在相同位点的基因敲除。这些结果将为恶性疟原虫的嘌呤途径提供明确的知识，关键步骤的遗传测试和针对该途径脆弱酶的强大过渡态抑制剂。 将通过追踪3H和14C标记的前体进入核酸，在培养的恶性疟原虫中定量嘌呤补救途径。将使用高灵敏度加速器质谱法（比计数灵敏度>106）来建立嘌呤补救通量，而不干扰嘌呤代谢物浓度。将在嘌呤核苷磷酸化酶（PNP）、HGXPRT、APRT和甲硫腺苷磷酸化酶（MTAP）的基因破坏中比较嘌呤补救通量。假设PNP和HGXPRT形成了优势嘌呤补救途径。该通路的消融可能诱导腺苷补救的次级通路。过渡态抑制剂将设计，合成和测试PNP和MTAP在这个实验室，和APRT和HGPRT（Grubmeyer实验室）。将测试过渡态抑制剂对红细胞中恶性疟原虫生长的影响，有和没有嘌呤补充剂以绕过代谢阻断。将通过比较基因敲除与过渡态抑制剂的代谢物掺入模式来评价途径阻断的有效性。验证嘌呤补救途径中的重要步骤将指导抑制剂设计的这一点和未来的计划。在嘌呤补救的关键步骤中诱导抑制剂阻断可能提供杀死疟原虫的有效策略。

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.