Dealing with Antibiotic Resistance: Antisense Technology

描述(申请人提供)：抗菌素耐药性是一个日益严重的问题，威胁着传染病的治疗和许多医疗程序。众所周知，新抗生素的引入是缓慢和昂贵的。因此，这项建议集中在与细菌耐药性问题作斗争的一个关键方面：寻找旨在保持现有抗生素有效性的策略 毒品。我们的模型系统是氨基糖苷6‘-N-乙酰转移酶Ib型[Aac(6’)-Ib]，它是革兰氏阴性病原菌中临床上最相关的乙酰基转移酶，它介导了对阿米卡星(AK)和其他氨基糖苷类药物的耐药性。这些抗生素是预防由几种革兰氏阴性病原体引起的严重感染的重要组成部分。这项研究的长期目标是开发与AK结合的药理工具，以克服AAC(6‘)-Ib的存在，并可成功地用于治疗对AK耐药的感染。我们已经确定了被称为外部引导序列(EGSS)的寡核苷酸序列，它们分别诱导RNaseP介导的AAC(6‘)-Ib和FtsZ mRNA的切割，并导致对AK的抗性水平的降低和细胞分裂的抑制。此外，我们还确定了由锁定核酸和脱氧核糖核苷酸单体(LNA/DNA)组成的抗核酸酶寡核苷酸类似物的行为类似于EGSS。该项目的目的之一是获得能够穿透细胞并抑制抗性基因aac(6‘)-Ib表达的高效LNA/DNA egss。这些化合物将与AK(LNA/DNA EGSaac/AK)结合使用，以实现对AK敏感性的表型转换。然而，由于反义策略的一个常见问题是抑制活性不足以达到有效的抗菌效果，因此我们将设计与LNA/DNA、EGSaac/AK混合物协同作用的化合物。我们将确定AAC(6‘)-Ib的抑制剂，它将消除由抗性基因残留表达产生的任何酶的活性。我们还将设计LNA/DNA egss，通过诱导FtsZ基因的RNase P-裂解来干扰大肠杆菌和鲍曼不动杆菌基本细胞分裂蛋白FtsZ的表达。将测试由LNA/DNA EGSaac/AK加AAC(6‘)-Ib抑制剂和/或靶向FtsZ的LNA/DNA EGS组成的组合，以确定它们抑制携带AAC(6’)-Ib的大肠杆菌和鲍曼不动杆菌的生长能力。

DESCRIPTION (provided by applicant): Antimicrobial resistance is a growing problem that threatens treatment of infectious diseases and numerous medical procedures. It is well known that the introduction of new antibiotics is slow and costly. In consequence, this proposal concentrates on a critical aspect of the fight against the problem of bacterial resistance to antibiotics: the search for strategies aimed at preserving the effectiveness of currently available drugs. Our model system is the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib], the most clinically relevant acetyltransferase among gram-negative pathogens, which mediates resistance to amikacin (Ak) and other aminoglycosides. These antibiotics are an important component of the armamentarium against serious infections caused by several gram-negative pathogens. The long term goal of this research is to develop pharmacological tools that in combination with Ak overcome the presence of aac(6')-Ib and can be successfully used to treat Ak-resistant infections. We have identified oligoribonucleotide sequences, known as external guide sequences (EGSs), that elicit RNase P-mediated cleavage of aac(6')-Ib and ftsZ mRNA and result in a reduction of the levels of resistance to Ak and inhibition of cell division, respectively. Furthermore, we determined that nuclease resistant oligoribonucleotide analogs composed of locked nucleic acids and deoxyribonucleotide monomers (LNA/DNA) behave as EGSs. One aim of this project is to generate efficient LNA/DNA EGSs that can penetrate the cells and inhibit expression of the resistance gene aac(6')-Ib. These compounds will be used in combination with Ak (LNA/DNA EGSaac/Ak) to achieve phenotypic conversion to susceptibility to Ak. However since a common problem of antisense strategies is that the inhibitory activity is not potent enough for an effective antimicrobial effect we will design compounds that act synergistically with the mix LNA/DNA EGSaac/Ak. We will identify inhibitors of AAC(6')-Ib that will eliminate the activity of any enzyme produced by residual expression of the resistance gene. We will also design LNA/DNA EGSs that will interfere with expression of the E. coli and A. baumannii essential cell division protein FtsZ by eliciting RNase P-cleavage of ftsZ mRNA genes. Combinations consisting of the mix LNA/DNA EGSaac/Ak plus an AAC(6')-Ib inhibitor and/or an LNA/DNA EGS targeting ftsZ will be tested to determine their ability to inhibit growth of E. coli and A. baumannii harboring aac(6')-Ib.