Novel Antifungals by Engineering the AbA NRPS gene

描述(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受损患者的数量持续增加，再加上30多年来只有一种新的抗真菌疗法被引入市场，因此迫切需要新的、更好的、具有新的作用模式(MOA)的抗真菌药物。天然产物Aureobasidin A(ABA)是一种有效的杀菌药物，具有一种新的MOA，也不会引起抗药性病原菌菌株。不幸的是，尽管有效且耐受性很好，但国产ABA的靶谱太窄，不能在临床上吸引人。在人类的两种主要病原体中，念珠菌属(Candida spp.)而曲霉菌、ABA对念珠菌只有抑制作用。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的ABA转化为对这两种病原体几乎同样有效的化合物。然而，所需的化学物质是复杂和昂贵的，因为它构成了阻碍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授予曲霉菌所需的结构修改。ABA的活性，从而避免了高昂的合成化学成本，并允许使用新型MOA实现有效、耐受性良好的抗真菌药物的商业化。在第一阶段，对编码非核糖体肽合成酶(NRPS)复合体的基因ABA 1进行了鉴定、克隆、测序和定位。到目前为止，第二阶段已经产生了方法和一套基因工具，允许高效地设计ABA 1基因。到目前为止，还成功地进行了ABA 1基因的工程设计，产生了产生结构修饰的ABA分子的工程菌，并产生了关于真菌NRPS复合体独特性质的重要新数据。通过设计真菌NRPS复合体来生产结构改变的环肽，以前还没有报道。到目前为止，该项目已经出版了两份出版物，一项已发布的专利和一项正在申请的专利。继续的第二阶段工作将涉及对授予曲霉菌种所需的具体修改进行工程设计。对ABA的活性和能够高产的产生菌的制备/选择。该项目的成功完成将：[1]向对新产品有强烈需求的市场提供一种高效、耐受性良好的药物；[2]满足目前几乎没有治疗选择的不断增长的患者群体的迫切需求；以及[3]为发现新的和改进的疗法的新的、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、癌症和其他免疫功能受损患者的数量持续增加，导致对具有新作用模式的新抗真菌药物的迫切需求尚未得到满足。拟议的项目将把一种有效、有效、耐受性好和经济的药物添加到目前有限且与重大限制相关的抗真菌药物清单中。

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.