Pathways to improved polyene antimicrobial agents (PIPA)

天然产物是由植物和微生物产生的分子，它们激发了我们今天所依赖的许多重要药物的开发。例如，土壤细菌，如放线菌科的链霉菌，在产生抗微生物剂方面特别多产，所述抗微生物剂可以杀死细菌、真菌和其他微生物病原体。这些天然抗菌剂中的许多已被广泛用于治疗危及生命的传染病。然而，现有的抗微生物药物由于抗微生物药物耐药性（AMR）而变得越来越无效，微生物病原体迅速进化出逃避这些化合物的影响的方法。真菌感染可能是特别成问题的，曲霉病和隐球菌病等疾病每年导致数百万例危及生命的感染。直到最近，才发现许多COVID-19患者（特别是在印度）死于由"黑真菌"毛霉菌病引起的继发感染。不幸的是，只有少量的抗真菌药物可用，并且很少有有希望的候选药物正在开发中。此外，目前使用的许多抗真菌药物对新出现的多重耐药真菌病原体（如耳念珠菌）基本无效，这些病原体对全球健康构成重大威胁。目前最有效和广泛使用的抗真菌剂是多烯阿替霉素B、制霉菌素和匹马菌素，它们都是世卫组织的基本药物。多烯类化合物是放线菌的天然产物，具有相似的大环结构。它们显示出优异的广谱抗真菌活性，并且与其他抗真菌药物不同，对多烯的耐药性不太普遍。尽管具有有利的性质，但多烯具有低溶解度并表现出毒性。这是因为多烯结合并破坏真菌病原体的细胞壁（膜），但也可以结合人类细胞膜，导致毒性作用。具有降低的毒性和增加的溶解度的多烯衍生物先前已经通过化学合成制备。然而，这通常需要费力且昂贵的多步合成程序，这些程序不可持续、污染严重，而且扩大生产成本太高。最近，我们对几种放射菌的基因组进行了测序，并发现了编码生物合成所需的酶（催化剂）的基因（组装）新型多烯。我们能够分离并确定新的多烯的结构。此外，我们获得了一些“剪裁”酶，在多烯生物合成的后期阶段增加关键功能的初步表征。在这个项目中，我们的目标是进一步研究新的剪裁酶，用天然底物（生物合成中间体）以及来自已知多烯的其他途径的前体（阿氏菌素B等）测试它们。我们还将获得结构或模型，我们可以用来突变（工程师）的剪裁酶，以扩大其底物范围，并促进替代剪裁反应的催化。然后，我们的目标是在反应中结合联合收割机不同的剪裁酶，以创建多烯衍生物的多样化库，将测试其抗真菌活性，毒性和溶解性。这将提供一个详细的结构-活性关系（SAR），使我们能够确定哪种剪裁反应的组合为多烯提供最佳性能。最初，我们将使用分离的酶（体外）生产新的多烯用于测试。然而，我们也将开发体内工程方法，以创建放线菌菌株，可以在一步发酵中产生最好的多烯衍生物。这些基于生物的方法，特别是体内发酵，可以为我们迫切需要的改进的多烯抗真菌剂提供更可持续，有效和具有成本效益的途径，以对抗新兴的耐药真菌病原体。

Natural products are molecules made by plants and microorganisms that have inspired the development of many important pharmaceuticals that we rely on today. For example, soil bacteria, such as Streptomyces from the Actinobacteria family, are particularly prolific in producing antimicrobial agents, which can kill bacterial, fungal, and other microbial pathogens. Many of these natural antimicrobial agents have been widely used to treat life-threatening infectious diseases. However, existing antimicrobial drugs are becoming increasingly ineffective due to antimicrobial resistance (AMR), with microbial pathogens rapidly evolving ways to evade the effects of these compounds. Fungal infections can be particularly problematic, with conditions such aspergillosis and cryptococcosis resulting in millions of life-threatening infections every year. Only very recently, it became apparent that many COVID-19 patients (particularly in India) died of a secondary infection, caused by the "black fungus" mucormycosis. Unfortunately, there are only a small number of antifungal drugs available and very few promising drug candidates in development. Moreover, many antifungals currently in use are largely ineffective against emerging multidrug-resistant fungal pathogens such as Candida auris which pose a major threat to global health. Currently the most effective and widely used antifungals are the polyenes amphotericin B, nystatin and pimaricin, which are all WHO essential medicines. Polyenes are complex natural products derived from Actinobacteria, with similar macrocyclic structures. They show excellent broad spectrum antifungal activity and, unlike other antifungal drugs, resistance to polyenes is less widespread. Despite possessing favourable properties, polyenes suffer from low solubility and exhibit toxicity. This is because polyenes bind to and disrupt the cell wall (membrane) of fungal pathogens, but can also bind to the human cell membrane, leading to toxic effects. Polyene derivatives with reduced toxicity and increased solubility have previously been prepared by chemical synthesis. However, this typically requires laborious and expensive multistep synthetic procedures, which are unsustainable, polluting and too costly to scale-up for manufacture.Recently, we have sequenced the genomes of several Actinobacteria and discovered genes encoding enzymes (catalysts) that are required for the biosynthesis (assembly) of novel polyenes. We were able to isolate and determine the structure of new polyenes. In addition, we obtained preliminary characterisation for some "tailoring" enzymes that add key functionality during the latter stages of polyene biosynthesis. In this project, we aim to further characterise the new tailoring enzymes, testing them with natural substrates (biosynthetic intermediates) as well as precursors from other pathways to known polyenes (amphotericin B etc.). We will also obtain structures or models which we can use to mutate (engineer) the tailoring enzymes to broaden their substrate scope and facilitate catalysis of alternative tailoring reactions. We then aim to combine the different tailoring enzymes in reactions to create a diverse library of polyene derivatives, which will be tested for antifungal activity, toxicity, and solubility. This will provide a detailed structure-activity relationship (SAR), enabling us to establish which combination of tailoring reactions provides polyenes with the best properties. Initially, we will use isolated enzymes (in vitro) to produce new polyenes for testing. However, we will also develop in vivo engineering approaches to create Actinobacterial strains that can produce the best polyene derivatives in a single-step fermentation. These bio-based approaches, particularly in vivo fermentation, can provide much more sustainable, efficient, and cost-effective routes to the improved polyene antifungal agents that we urgently need to combat the emerging drug-resistant fungal pathogens.