Methods for bioengineering NRPS/PKS assembly lines delivering peptide natural products with electrophilic warheads.

天然产物(NP)是从微生物和植物中分离出来的分子，它们启发了许多领先的抗生素、抗癌、免疫抑制剂和其他基本药物的发展，这些药物在今天的临床上被广泛使用。通常，从天然有机体中分离出来的NP一开始就不具备必要的特性。通常需要进一步的合成修饰才能提供最终的药物化合物。然而，NP是典型的高度复杂的分子，需要费力的多步骤化学合成，这是非常昂贵的，污染和越来越不可持续的。与合成优化的NP变体相关的困难是制药公司进行药物开发的主要障碍。这在生产治疗疟疾、利什曼病和恰加斯病等发展中国家疾病所需的药物方面尤其成问题。这些高度传染性疾病是由昆虫传播的单细胞(原生动物)寄生虫引起的，影响着世界上数十亿最贫穷的人，每年导致50多万人死亡。目前，针对这些疾病的有效治疗方法很少。一种NP青蒿素被用于治疗疟疾，但出现了对青蒿素具有抗药性的新的疟疾寄生虫株(恶性疟原虫)。已经确定了治疗疟疾和其他相关疾病的有希望的新NP先导，但合成衍生物的成本阻碍了新的治疗方法的出现。生产优化的NP衍生物的另一种选择是操纵构成母体NP的微生物中的生物合成装配线(酶)。通过重新编程(工程)装配线以接受不同的前体，具有改进特性的NP变体可以在更有效、更具成本效益的单步发酵过程中被提供。在这个项目中，我们的目标是设计生物合成途径来生产具有抗原虫活性的NP衍生物，可以用于对抗疟疾或相关疾病。靶标NP是由链霉菌和其他细菌产生的具有反应性末端官能团(弹头)的氨基酸组成的多肽。这些NP将被设计成与原生动物的蛋白酶体结合，如恶性疟原虫。蛋白酶体是一种大型多蛋白质复合体，负责降解细胞中受损或不再需要的其他蛋白质。多肽NP的弹头可以与蛋白酶体交联，抑制其功能，导致细胞死亡。我们将使用新的基因编辑和其他方法来设计编码酶的基因，这些酶组装含有多肽NP的弹头。这将使我们能够改变多肽的序列，还可以包括不同的弹头，以提高它们的活性、选择性和药物开发的其他性质。生物合成装配线包括缩合氨基酸前体的非核糖体肽合成酶(NRPS)酶。通过替换NRPS中的结构域或亚结构域，可以改变多肽产品中的氨基酸序列。在早期合成研究的指导下，我们将创造含有弹头的多肽，这些多肽对恶性疟原虫蛋白酶体具有高度选择性。抑制人类和寄生虫细胞中的蛋白酶体的化合物是有毒的，不适合使用。我们还将设计装配线，提供含有弹头的多肽，这些多肽旨在抑制人类癌细胞中的蛋白酶体。这包括一种合成类似物oprozomib，它正在进行临床试验，用于治疗多发性骨髓瘤(骨髓癌)。通过开发一种获得这种类型化合物的工程方法，有可能在一步发酵中生产出像oprozomib这样的抗癌药物，使它们以更低的成本更广泛地获得。我们开发的方法是通用的，可以用于生产一系列含有多肽的弹头，用于许多其他治疗应用。

Natural products (NP) are molecules isolated from microorganisms and plants that inspired the development of many leading antibiotics, anticancer, immunosuppressive agents and other essential medicines that are widely used in the clinic today. Often the NP that are isolated from the native organism do not possess the prerequisite properties at the outset. Further synthetic modification is typically required to provide the final drug compound. However, NP are typically highly complex molecules requiring laborious multistep chemical synthesis, which is very expensive, polluting and increasingly unsustainable. The difficulty associated with the synthesis of optimised NP variants presents a major barrier to pharma companies undertaking drug development. This is particularly problematic in the manufacture of drugs required to treat diseases of developing world such as malaria, Leishmania and Chagas disease. These highly infectious diseases, caused by single celled (protozoan) parasites transmitted by insects, effect billions of the poorest people in the world and lead to over 500,000 deaths pa. Currently there are very few effective treatments available for these diseases. A NP artemisinin is used to treat malaria, but new strains of the malaria parasite (P. falciparum) have emerged which are resistant to artemisinin. Promising new NP leads have been identified for malaria and other related diseases, but the costs of synthesising derivatives have prevented new treatments being made available. An alternative for producing optimised NP derivatives, is to manipulate the biosynthetic assembly lines (enzymes) in the microorganisms that construct the parent NP. By reprogramming (engineering) the assembly line to accept different precursors, NP variants with improved properties can be delivered in a more efficient, cost-effective single-step fermentation process.In this project we aim to engineer biosynthetic pathways to produce NP derivatives with antiprotozoal activity that could be used to combat malaria or related diseases. The target NP are peptides, composed of amino acids with a reactive terminal functional group (warhead), produced by Streptomyces and other bacteria. These NP will be designed to bind to the proteasome of protozoa such as P. falciparum. Proteasomes are large multi-protein complexes responsible for degrading other proteins in the cell that are either damaged or no longer needed. The warhead of the peptide NP can cross-link with the proteasome inhibiting its function leading to cell death. We will use novel gene editing and other approaches to engineer the genes encoding the enzymes that assemble the warhead containing peptide NP. This will allow us to change the sequence of the peptides and also include different warheads, to improve their activity, selectivity and other properties for drug development. The biosynthetic assembly line includes nonribosomal peptide synthetase (NRPS) enzymes that condense amino acid precursors. By replacing domains, or subdomains, within the NRPS it is possible to change the sequence of the amino acids in the peptide products. Guided by earlier synthetic studies, we will create warhead containing peptides that are highly selective for the P. falciparum proteasome. Compounds that inhibit proteasomes in human as well as the parasite cell, would be toxic and unsuitable. We will also engineer assembly lines that deliver warhead containing peptides designed to inhibit the proteasomes in human cancer cells. This includes oprozomib a synthetic analogue, which is in clinical trials for treatment of multiple myeloma (bone marrow cancer). By developing an engineered pathway to this type compound, it may be possible to produce anticancer drugs, like oprozomib, in a single-step fermentation making them more widely available at lower costs. The methods we develop are generic and can be used to produce a range of warhead containing peptides for a number of other therapeutic applications.