Novel Antifungals by Engineering the AbA NRPS gene

描述（由申请人提供）：需要治疗真菌感染的手术、移植、癌症和其他免疫功能低下患者数量的持续增加，加上事实上只有一类新的抗真菌治疗药物被引入市场。30多年来，人们迫切需要具有新型作用方式（MoA）的新的、更好的抗真菌药物。 天然产物化合物Aureobasidin A（阿坝）是一种有效的杀真菌药物，具有新的MoA，也不会引起抗性病原体菌株。不幸的是，尽管天然阿坝有效且耐受性非常好，但其靶谱太窄而不具有临床吸引力。在两种主要的人类病原体中，念珠菌属（Candida spp.）和曲霉属，阿坝A仅对念珠菌有效。然而，探索性合成化学工作已经证明，结构修饰可以将天然阿坝转化为对两种病原体具有接近相等功效的化合物。然而，所需的化学过程是复杂和昂贵的，以至于它构成了将这些化合物开发成商业产品的障碍。 本提案中概述的项目的总体目标是使用一种新的基因工程方法来引入赋予曲霉菌所需的结构修饰。本发明提供了一种具有抗阿坝活性的抗真菌药物，从而避免了合成化学的高成本，并允许具有新MoA的有效的、耐受性良好的抗真菌药物的商业化。 在第一阶段，基因，aba 1，编码非核糖体肽合成酶（NRPS）复合物，负责合成的阿坝在生产生物体进行了鉴定，克隆，测序和定位。第二阶段迄今已产生的方法和一套遗传工具，使有效的工程的阿坝1基因。迄今为止，还完成了aba 1基因的成功工程改造，产生了产生结构修饰的阿坝分子的工程菌株，并产生了关于真菌NRPS复合物独特性质的重要新数据。通过真菌NRPS复合物的工程化来生产结构改变的环肽先前尚未报道。迄今为止，该项目已出版了两份出版物，一份已获专利，一份正在申请专利。继续的第二阶段工作将涉及赋予曲霉属所需的特定修饰的工程。对阿坝的活性和能够高生产水平的生产菌株的制备/选择。 该项目的成功完成将：[1]为对新产品有强烈需求的市场提供一种有效，耐受性良好的药物; [2]解决目前治疗选择很少的日益增长的患者群体的迫切需求;[3]为发现新的和改进的治疗方法的新的和潜在的非常强大的方法提供概念证明和关键工具。 公共卫生相关性：需要治疗真菌感染的手术、移植、癌症和其他免疫受损患者数量的持续增加，已经产生了对具有新作用模式的新抗真菌药物的未满足的需求。拟议的项目将增加一个强大的，有效的，耐受性良好和经济的药物库存的抗真菌药物，目前是有限的，并与显着的局限性。

DESCRIPTION (provided by applicant): The continuing increase in the number of surgery, transplantation, cancer and other immunocompromized patients, that need treatment for fungal infections, together with the fact that only one new class of antifungal therapeutics has been introduced to the market in over 30 years has created an immediate need for new and better antifungal drugs with novel modes of action (MoA). The natural product compound Aureobasidin A (AbA) is a potent, fungicidal drug with a novel MoA that also does not elicit resistant pathogen strains. Unfortunately, although efficacious and very well tolerated, native AbA's target spectrum is too narrow to be clinically attractive. Of the two major human pathogens, Candida spp. and Aspergillus spp., AbA only has efficacy against Candida. However, exploratory synthetic chemistry work has demonstrated that structural modifications can convert native AbA into compounds that have close to equal efficacy against both pathogens. The required chemistry, however, is complicated and expensive, to the extent that it constitutes a barrier against development of these compounds into commercial products. The overall goal of the project outlined in this proposal is to use a novel genetic engineering approach to introduce the structural modifications required to confer Aspergillus spp. activity to AbA, thereby avoiding the high cost of synthetic chemistry and allow commercialization of an efficacious, well tolerated antifungal drug with a novel MoA. In Phase I, the gene, aba 1, encoding the non-ribosomal peptide synthetase (NRPS) complex responsible for synthesis of AbA in the producer organism was identified, cloned, sequenced and mapped. Phase II has to date produced methodologies and a set of genetic tools that allow efficient engineering of the aba 1 gene. Also accomplished to date is the successful engineering of the aba 1 gene, the generation of engineered strains producing structurally modified AbA molecules and the generation of significant new data on the unique properties of fungal NRPS complexes. Production of structurally altered cyclic peptides by engineering of a fungal NRPS complex has not been reported previously. The project has to date produced two publications, one issued patent and one pending patent application. The continued Phase II work will involve engineering of the specific modifications required to confer Aspergillus spp. activity to AbA and the preparation/selection of a producer strain capable of high production levels. Successful completion of the project will: [1] provide an efficient, well-tolerated drug to a market with a strong demand for new products; [2] address a very immediate need from a growing patient population which currently have very few treatment options; and [3] provide proof of concept and critical tools for a novel and potentially very powerful approach to the discovery of new and improved therapeutics. PUBLIC HEALTH RELEVANCE: The continuing increase in the number of surgery, transplantation, cancer and other immunocompromized patients, that need treatment for fungal infections, has generated an immediate unmet need for new antifungal drugs with novel modes of action. The proposed project will add a potent, efficacious, well-tolerated and economical drug to an inventory of antifungal drugs that currently is both limited and associated with significant limitations.