Generation of recombinant thiopeptides to target antimicrobial-resistant bacteria

描述（由申请人提供）：拟议项目的长期目标是通过将重组DNA技术的力量与天然产物抗生素的生物合成相结合，开发针对耐药细菌病原体的连续和遗传编码的新抗生素来源。随着危及生命的耐药细菌如耐甲氧西林金黄色葡萄球菌（MRSA）和耐万古霉素肠球菌（VRE）的出现，对新抗生素的需求比以往任何时候都大。然而，新抗生素的开发近50年来一直停滞不前。一种有前途的新型天然产物，称为硫肽，在低纳摩尔浓度下抑制这些革兰氏阳性细菌的生长。硫肽作为潜在的抗生素是令人兴奋的，因为它们的作用位点，核糖体蛋白L11和23S rRNA之间的界面，与所有现有的抗生素类别不同。重要的是，这些天然产物来源于遗传编码的肽，其允许通过简单诱变产生新的硫肽类似物。我们选择研究硫肽thiocillin作为模型系统，因为与其他硫肽不同，thiocillin是在遗传上易处理的蜡状芽孢杆菌菌株中产生的，并且已知其耐受各种突变。该项目的近期目标是设计一种方法，快速多样化和筛选这些硫肽天然产物，从而加速新抗生素的发现。为了实现这一目标，我们将测试的假设，硫代青霉素的诱变将提供一个广阔的景观的结构和化学多样性，能够针对耐药细菌。该项目的R21阶段将专注于开发新的重组方法，以快速产生大量的硫代西林天然产物库，并筛选新的抗生素。R33阶段将开发一种针对潜在耐药突变的方法，并通过药物开发管道获得有希望的成功。药物开发阶段的重点是增加硫西林的溶解度，以开发用于治疗全身性细菌感染的IV制剂。如果成功，项目 将对公共卫生产生重大影响，因为开发新的抗生素药物对于应对日益严重的新出现的耐药性问题是必要的。此外，它将为产生一种新的多样化的天然产品类别奠定基础，以筛选许多其他治疗应用。

DESCRIPTION (provided by applicant): The long-term goal of the proposed project is to develop a continuous and genetically encoded source of new antibiotics against drug-resistant bacterial pathogens by combining the power of recombinant DNA technology with the biosynthesis of natural product antibiotics. With the emergence of life-threatening drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), the need for new antibiotics is greater than ever. Yet, the development of new antibiotics has remained stagnant for nearly 50 years. A promising new class of natural products, called thiopeptides, inhibits the growth of these gram-positive bacteria at low nanomolar concentrations. Thiopeptides are exciting as potential antibiotics, because their site of action, the interface between ribosomal protein L11 and 23S rRNA, is distinct from all existing classes of antibiotics. Importantly, these natural products are derived from genetically encoded peptides, which allow for the generation of new thiopeptide analogs by simple mutagenesis. We have chosen to study the thiopeptide thiocillin as a model system, because unlike other thiopeptides, thiocillin is produced in the genetically tractable Bacillus cereus strain and is known to tolerate a variety of mutations. The immediate goal of the proposed project is to devise a method that will rapidly diversify and screen these thiopeptide natural products and thus accelerate discovery of new antibiotics. To achieve this goal, we will test the hypothesis that mutagenesis of thiocillin will provide a vast landscape of structural and chemical diversity capable of targeting antibiotic-resistant bacteria. The R21 phase of this project will focus on developing novel recombinant methods to rapidly generate large libraries of thiocillin natural products and to screen for new antibiotics. The R33 phase will develop a method to target potential resistance mutations and take promising hits through the drug development pipeline. The drug development phase will focus on increasing the solubility of thiocillin to develop an IV formulation for the treatment of systemic bacterial infections. If successful, the proposed project will have a significant impact on public health because the development of new antibiotic drugs is necessary to combat the rising problem of emerging drug resistance. Moreover, it will lay the groundwork for generating a new diverse class of natural products to screen for many other therapeutic applications.