Chemoenzymatic studies of aminoglycoside-resistance enzymes towards new drugs

说明(申请人提供)：氨基糖苷类抗生素是一种广谱抗生素，用于治疗严重的细菌感染，包括致命的结核病和伴随艾滋病、囊性纤维化和癌症的细菌感染。对这些药物具有抗药性的病原体的出现是对公共健康的重大威胁，并突出表明需要新的抗菌剂。在目标1中，我们建议利用氨基糖苷乙酰转移酶(AAC)家族的氨基糖苷类修饰酶，结合新开发的6‘-N-酰化保护基团的化学方法学(I)建立新的和更有效的N-酰化氨基糖苷类抗生素的体外文库，以及(Ii)开发氨基糖苷类探针，作为诱饵来鉴定这些抗生素的新的治疗蛋白质靶点/途径。我们的化学酶和化学策略为以下问题提供了有效的解决方案：(I)目前还没有通用的合成方法来创建N-酰化氨基糖苷类化合物，以及(Ii)没有有效的方法来化学修饰含有一系列化学相同胺的氨基糖苷类化合物上的特定胺基。现有的几个仅使用化学合成的例子对研究时间和成本要求太高，而且仅限于非常具体的情况。在目标2中，我们建议对广泛耐药结核分枝杆菌(XDR-TB)氨基糖苷耐药的一个主要决定因素的作用机制和抑制机制进行生化和结构研究。我们希望这项工作(I)促进对包括结核分枝杆菌在内的各种病原菌对AG耐药性的基本了解，并(Ii)提供一种潜在的解决方案，以克服大多数XDR-TB的氨基糖苷类耐药性问题。与公众健康相关：我们预计这项工作将有助于开发新的抗生素，有可能对抗现有的和新出现的耐药细菌。 公共卫生相关性：拟议的研究与公共健康相关，因为对氨基糖苷类耐药酶的更好理解有望导致新药的发现和开发，并有助于确定药物靶点。这项研究将使我们能够更好地处理感染细菌对氨基糖苷类抗生素的耐药性问题。这项拟议的研究将在两个方面产生重大影响：(1)我们的新的化学和化学酶方法用于氨基糖苷类抗生素的快速和简便的区域选择性N-酰化反应，将大大降低与此类修饰相关的成本和工作量；(2)我们对广泛耐药结核分枝杆菌氨基糖苷耐药主要决定因素的作用机制和抑制机制的研究将促进我们对耐药性的理解，并为抗生素领域建立氨基糖苷乙酰转移酶的新范式。

DESCRIPTION (provided by applicant): Aminoglycosides are broad-spectrum antibiotics used for treatment of serious bacterial infections, including the deadly tuberculosis and those accompanying AIDS, cystic fibrosis, and cancer. The emergence of pathogens resistant to these drugs represents a major threat to public health and underscores the need for new antimicrobial agents. In Aim 1, we propose to utilize aminoglycoside-modifying enzymes of the aminoglycoside acetyltransferase (AAC) family in conjunction with a newly developed 6'-N-acylation protecting group-free chemical methodology (i) to generate in vitro libraries of new and more potent N-acylated aminoglycoside antibiotics, and (ii) to develop aminoglycoside probes that will serve as baits for identification of novel therapeutic protein targets/pathways for these antibiotics. Our chemoenzymatic and chemical strategies offer an effective solution to the following problems: (i) there are no existing general synthetic methodologies for the creation of N-acylated aminoglycosides, and (ii) there are no efficient methods to chemically modify specific amine groups on an aminoglycoside that contains a series of chemically identical amines. A few existing examples that use solely chemical syntheses are too demanding on research time and cost and are limited to very specific cases. In Aim 2, we propose biochemical and structural studies of the mechanism of action and inhibition of a major determinant of aminoglycoside resistance in extensively drug-resistant strains of M. tuberculosis (XDR-TB). We expect this work to (i) advance the basic understanding of AG resistance of a variety of pathogenic bacteria, including M. tuberculosis, and (ii) provide a potential solution to overcome the aminoglycoside resistance problem in majority of XDR- TB. Relevance to public health: We expect that this work will contribute to the development of novel antibiotics with a potential to combat existing and newly emerging drug-resistant bacteria. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health as an improved understanding of aminoglycoside- resistance enzymes is expected to lead to the discovery and development of new drugs and also in identifying drug targets. This research will enable us to better handle the problem of resistance of infectious bacteria to aminoglycoside antibiotics. The proposed research will have a major impact in two ways: (1) our novel chemical and chemoenzymatic methodologies for the rapid and facile regio- selective N-acylation of aminoglycosides will greatly reduce the cost and effort generally associated with such modifications, and (2) our study of the mechanism of action and inhibition of the major determinant of aminoglycosdie resistance in extensively drug-resistant M. tuberculosis will advance our understanding of drug resistance and also set a new paradigm of aminoglycoside acetyltransferase in the antibiotics field.