EF-Tu binding Tetrazoles targeting MDR Neisseria gonorrhoeae

项目摘要 CDC将耐多药淋病奈瑟菌（Ng）列为三大最紧迫的抗生素耐药性威胁之一 在美国作为一种革兰氏阴性苛养微生物，Ng引起淋病， 性传播细菌感染（STI），在美国每年估计有> 800，000例。左 如果不治疗，淋病会导致女性盆腔炎，导致输卵管疤痕， 不育或可能传播，引起关节和皮肤的表现。曾经很容易治疗的Ng已经进化成 对几乎每一种用于治疗它的抗生素都有耐药性，留下了阿奇霉素（AZM）和 头孢曲松（CTX）是目前唯一可用的治疗选择。重要的是，最近有一组病例 在夏威夷报道了一种对AZM和CTX都有耐药性的病毒，这突出了对新疗法的迫切需求 针对抗生素耐药性Ng感染。 细菌翻译受到转录错误、mRNA损伤和翻译移码的困扰， 可导致终止密码子的丢失和非终止核糖体复合物的形成，从而阻止 蛋白质产物并抑制进一步翻译。恢复不间断的核糖体复合物是一个必不可少的 在细菌中的翻译过程中，每一个细菌基因组中都有反式翻译机制， 约会反式翻译组分中的突变影响多种细菌的生存力或毒力 包括吴 在初步研究中，我们证明了基于四唑的化合物抑制反式翻译， 针对一系列病原体（包括Ng）的强效抗菌剂，关键类似物抑制>95%的Ng 在极低浓度下检测的菌株（MIC 90 = 0.27 μg/mL）。光亲和标记和生物化学 实验表明，反式翻译的抑制是通过化合物结合到一个新的位点介导的， 延伸因子Tu（EF-Tu;蛋白质合成和反式翻译的重要组成部分）作用于 选择性地反对反译而不是翻译。对这些化合物的初步评价表明， 有限的细胞毒性和响应性SAR，能够优化效力并导致鉴定 MBX-3808，该提案中的主要化合物。 本I期申请的目的是证明这些反式翻译抑制剂是有效的 在淋病感染的动物模型中，并建立一个数据集，将推动从命中到导致的过渡， 电极导线优化。第二阶段将重点关注先导化合物的优化，以生产适合IND的临床前候选药物。 赋能研究。我们将通过四个具体目标实现第一阶段的目标： 化学优化反式翻译抑制剂系列的效力、选择性和体外ADME性质， 靶向化学合成>200个类似物。在目标2中，我们将鉴定具有ADME-T和效力的类似物 适用于体内测试、分析效力和体外特性的所有材料， 目标1。目的3：我们将证实并进一步探讨四唑类药物抑制剂的作用机制和特异性。 在Ng.在目标4中，我们将评估铅类似物的生物利用度、急性毒性和功效 Ng感染的鼠模型。

Project Summary The CDC lists MDR Neisseria gonorrhoeae (Ng) as one of the three most urgent antibiotic resistance threats in the United States. A Gram-negative fastidious organism, Ng causes gonorrhea, the second-most prevalent sexually transmitted bacterial infection (STI) with >800,000 estimated cases in the United States annually. Left untreated, gonorrhea can cause pelvic inflammatory disease in women, leading to fallopian tube scarring and infertility or may disseminate, causing joint and skin manifestations. Once easily treatable, Ng has evolved resistance to nearly every antibiotic used to treat it, leaving a combination of azithromycin (AZM) and ceftriaxone (CTX) as the only currently available treatment option. Importantly, a cluster of cases was recently reported in Hawaii that was resistant to both AZM and CTX, highlighting the critical need for new therapeutics targeting antibiotic-resistant Ng infections. Bacterial translation is plagued by transcription errors, mRNA damage, and translational frameshifts, which can result in loss of the stop codon and non-stop ribosome complex formation, preventing the release of protein products and inhibiting further translation. Recovery of non-stop ribosome complexes is an essential process in bacteria mediated by trans-translation machinery found in every bacterial genome sequenced to- date. Mutations in trans-translation components affect viability or virulence in a wide variety of bacteria including Ng. In preliminary studies, we demonstrated that tetrazole-based compounds inhibit trans-translation, acting as potent antimicrobials against a range of pathogens, including Ng, with a key analog inhibiting >95% of Ng strains tested at extremely low concentrations (MIC90 = 0.27 μg/mL). Photoaffinity labeling and biochemical experiments suggest that inhibition of trans-translation is mediated through compound binding to a novel site of Elongation factor Tu (EF-Tu; an essential component of protein synthesis and trans-translation) acting selectively against trans-translation over translation. Preliminary evaluation of these compounds demonstrated limited cytotoxicity and a responsive SAR, enabling optimization of potency and resulting in the identification of MBX-3808, the lead compound in this proposal. The objective of this Phase I application is to demonstrate that these trans-translation inhibitors are effective in animal models of gonorrhea infection and build a dataset that will drive the transition from Hit-to-Lead to Lead Optimization. Phase II will focus on lead optimization to produce a preclinical candidate suitable for IND- enabling studies. We will achieve the Phase I objectives through four specific aims: In Aim 1 we will chemically optimize the trans-translation inhibitor series for potency, selectivity and in vitro ADME properties, targeting chemical synthesis of >200 analogs. In Aim 2, we will identify analogs with ADME-T and potency properties suitable for in vivo testing, analyzing potency and in vitro properties for all materials generated in Aim 1. In Aim 3, we will confirm and further explore the mechanism and specificity of tetrazole inhibitor of trans-translation in Ng. In Aim 4, we will evaluate lead analogs for bioavailability, acute toxicity and efficacy in murine models of Ng infection.