Dealing with Antibiotic Resistance: Antisense Technology

描述（由申请人提供）：抗生素耐药性是一个日益严重的问题，威胁着感染性疾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，本提案集中于解决细菌对抗生素耐药性问题的一个关键方面：寻求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]，其是革兰氏阴性病原体中最临床相关的乙酰转移酶，其介导对阿米卡星（Ak）和其他氨基糖苷类的耐药性。这些抗生素是治疗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。本研究的长期目标是开发与Ak组合克服aac（6 '）-Ib的存在并可成功用于治疗Ak-resistant感染的药理学工具。我们已经鉴定了称为外部引导序列（EGS）的寡核糖核苷酸序列，其引起RNase P介导的aac（6 '）-Ib和ftsZ mRNA的切割，并分别导致对Ak的抗性水平降低和细胞分裂抑制。此外，我们确定了由锁核酸和脱氧核糖核苷酸单体（LNA/DNA）组成的核酸酶抗性寡核糖核苷酸类似物表现为EGS。该项目的一个目的是产生有效的LNA/DNA EGSs，其可以穿透细胞并抑制抗性基因aac（6 '）-Ib的表达。这些化合物将与Ak（LNA/DNA EGSaac/Ak）联合使用，以实现表型转化为对Ak的敏感性。然而，由于反义策略的一个共同问题是抑制活性对于有效的抗微生物作用来说不够强，我们将设计与LNA/DNA EGSaac/Ak混合物协同作用的化合物。我们将鉴定AAC（6 '）-Ib的抑制剂，其将消除由抗性基因的残余表达产生的任何酶的活性。我们还将设计LNA/DNA EGSs，其将干扰E. coli和A.通过引发ftsZ mRNA基因的RNase P-切割来表达鲍曼不动杆菌必需细胞分裂蛋白FtsZ。将测试由LNA/DNA EGSaac/Ak混合物加上AAC（6 ′）-Ib抑制剂和/或靶向ftsZ的LNA/DNA EGS组成的组合，以确定它们抑制大肠杆菌生长的能力。coli和A.鲍曼不动杆菌含有aac（6 '）-lb。

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.