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30多年来只有一种新型抗真菌治疗药物被引入市场，这就产生了对具有新型作用模式（MoA）的新型更好的抗真菌药物的迫切需求。天然产物化合物Aureobasidin A （AbA）是一种有效的杀真菌药物，具有一种新的MoA，也不会引起耐药病原体菌株。不幸的是，尽管天然AbA有效且耐受性良好，但其靶标谱太窄，在临床上缺乏吸引力。在两种主要的人类病原体念珠菌和曲霉中，AbA仅对念珠菌有效。然而，探索性的合成化学工作已经证明，结构修饰可以将天然AbA转化为对这两种病原体具有接近同等功效的化合物。然而，所需的化学反应是复杂和昂贵的，在某种程度上，它构成了阻碍这些化合物发展成商业产品的障碍。本项目概述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属AbA活性所需的结构修饰，从而避免合成化学的高成本，并使具有新型MoA的有效，耐受性良好的抗真菌药物商业化。在第一阶段，研究人员鉴定、克隆、测序和定位了产生生物体内负责aba合成的非核糖体肽合成酶（NRPS）复合物的编码基因aba 1。到目前为止，第二阶段已经产生了方法和一套遗传工具，使aba 1基因的有效工程。迄今为止，aba 1基因的成功工程，产生具有结构修饰的aba分子的工程菌株，以及关于真菌NRPS复合物独特性质的重要新数据的产生也取得了成功。通过真菌NRPS复合物工程生产结构改变的环肽以前没有报道。该项目迄今已出版了两份出版物，一份已颁发的专利和一份正在申请的专利。继续进行的第二阶段工作将包括工程上的特定修改，以赋予曲霉属的AbA活性，并准备/选择能够高生产水平的生产菌株。该项目的成功完成将：[1]为新产品需求旺盛的市场提供一种高效、耐受性好的药物；[2]解决了日益增长的患者群体的迫切需求，这些患者目前的治疗选择非常少；[3]提供了概念证明和关键工具，为发现新的和改进的治疗方法提供了一种新颖的、潜在的非常强大的方法。

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.