Peptide Deformylase: Mechanism and Inhibitor Design

描述(申请人提供)：本项目的目标是(1) 去甲酰化N-甲酰化肽(肽)的酶的特性 脱甲酰基酶(PDF)、甲硫氨酸氨基肽酶(FMAP)和甲酰蛋氨酸 脱甲酰基酶(FMDF))，以及(2)评估PDF作为新的抗菌靶标 药物设计。在细菌和真核细胞器中，蛋白质合成启动 N-甲酰蛋氨酸。因此，所有新合成的多肽都在 细菌和细胞器带有N-末端甲酰基。跟随 翻译启动，PDF从绝大多数分子中去除了N-甲酰基 细菌蛋白质。作为细菌中的一种基本活动，PDF正在被 作为抗菌药物设计的新靶点。最近，基因组学 测序已经在某些真核生物中发现了类似PDF的序列， 对PDF作为药物靶点的有效性提出了一些问题。A相关 问题是用PDF抑制剂治疗是否会导致蓄积 N-甲酰基多肽与患者炎症的关系，因为一些N-甲酰基 多肽(例如，f-MLF)是有效的趋化剂。为了解决这些问题， 本项目提出了五个具体目标。具体目标1是执行 大肠杆菌PDF结构与功能关系的定量分析。 活性部位的保守残基将发生突变， 并进行结构表征。具体目标2是克隆 并对人和恶性疟原虫的PDF类序列进行了特征分析， 疟疾的病原体。我们的目标是确定这些 序列实际上编码了功能性PDF及其生理功能。 具体目标3是进一步提高PDF的效力和特异性 针对细菌PDF的抑制剂，设计有效的抗P. 恶性疟原虫PDF，并评价PDF作为抗疟疾的潜在靶点 药物设计。具体目标4是设计能够选择性地 由PDF激活。这类前药预计会更具特异性(因此较少 有毒)，细菌很难产生抗药性。具体目标5是 纯化、克隆和鉴定大鼠FMAP和FMDF。这些酶是 被认为负责灭活由 共生细菌和防止哺乳动物的炎症反应。

DESCRIPTION (provided by the applicant): The objective of this project is (1) to characterize the enzymes which deformylate N-formylated peptides (peptide deformylase (PDF), formylmethionine aminopeptidase (fMAP), and formylmethionine deformylase (fMDF)), and (2) to assess PDF as a novel target for antimicrobial drug design. In bacteria and eukaryotic organelles, protein synthesis initiates with N-formylmethionine. Consequently, all newly synthesized polypeptides in bacteria and organelles carry an N-terminal formyl group. Following translational initiation, PDF removes the N-formyl group from the vast majority of bacterial proteins. As an essential activity in bacteria, PDF is being pursued as a new target for antibacterial drug design. Recently, genomic sequencing has revealed PDF-like sequences in certain eukaryotes including man, raising some questions about the validity of PDF as a drug target. A related issue is whether treatment with PDF inhibitors would result in the accumulation of N-formyl peptides and inflammation in a patient, since some of the N-formyl peptides (e.g., f-MLF) are potent chemotactic agents. To address these issues, five specific aims are proposed in this project. Specific Aim 1 is to perform quantitative analyses of the structure-function relationship of E. coil PDF. Conserved residues in the active site will be mutated and kinetic, spectral, and structural characterization will be conducted. Specific Aim 2 is to clone and characterize the PDF-like sequences from human and Plasmodium falciparum, the causative agent of malaria. The goal is to determine whether these sequences actually code for functional PDF's and their physiological functions. Specific Aim 3 is to further improve the potency and specificity of PDF inhibitors against bacterial PDF, to design potent inhibitors against P. falciparum PDF, and to evaluate PDF as a potential target for antimalarial drug design. Specific Aim 4 is to design prodrugs that can be selectively activated by PDF. Such prodrugs are expected to be more specific (thus less toxic) and difficult for bacteria to develop resistance. Specific Aim 5 is to purify, clone, and characterize fMAP and fMDF from rats. These enzymes are thought to be responsible for inactivating the chemotactic peptides released by commensal bacteria and preventing inflammatory responses in mammals.