Discovery of Novel Nontuberculous Inhibitors

项目摘要/摘要 非结核分枝杆菌(Ntm)是广泛存在于环境中的病原体和致病菌。 进行性肺部疾病以及皮肤和软组织、中枢神经系统和播散性感染。 NTM感染率正在全球范围内上升，并已成为全球重要的人类病原体。这个 引起这些感染的特定病原体属于以下两种之一：i)脓肿分枝杆菌 (M.脓肿)复合体(MABSC)和II)禽分枝杆菌(M.avium)复合体。脓肿分枝杆菌 由脓肿分枝杆菌、马氏分枝杆菌和博莱蒂分枝杆菌亚种组成的复合体已经成为一个重要的 全球威胁导致结构性肺患者肺部感染数量增加 慢性阻塞性肺病、支气管扩张症和囊性纤维化等疾病。特别的 值得关注的是，合并MABSC病原体感染的CF患者通常无法治疗，尽管多年来 联合治疗导致60%-70%的治疗失败。因此，新型抗非结核分枝杆菌制剂具有很强的药效 对MABSC菌株的活性可以缩短治疗时间，降低耐药率，并提高 我们迫切需要药效。 我们发现了一系列新的吲哚-2-羧胺(IC)，它们具有最低限度的有效抑制作用 对各种临床上生长缓慢和快速生长的NTM的最低抑菌浓度(MIC值)为0.0039-8微克/毫升， 包括脓肿分枝杆菌、马氏分枝杆菌、博莱蒂分枝杆菌和龟分枝杆菌。此外，IC在体外是有效的， 在小鼠身上，展示了它们的安全和有效的轮廓。然而，IC是亲脂性的，不容易溶于水 导致口腔吸收不良。为了避免这种不良的药代动力学特性，我们建议设计 新型氨基酸IC类似物和载药脂质体的开发是一种安全有效的选择 配制难溶的IC。将评估新的类似物的抗分枝杆菌活性和活性 化合物将接受一系列ADMETox分析，以确定可能的PK/PD作用。IC和IC模拟 装载的脂质体将被设计为吸入型给药，并被设计成优先被吸收 通过巨噬细胞，NTM病原体居住在感染的患者体内。这将通过以下方式实现 ICs的合成、载药脂质体的制备及药物释放和巨噬细胞的表征 领悟。脂质体将使用薄膜水合方法生产，然后使用IC加载 被动诱捕方法。脂质体的片层将通过31P核磁共振进行监测，并与 低温透射电子显微镜。将进行药物释放研究，以确保释放治疗IC浓度 (浓度高于MIC值)。最后，优化药物释放的IC载脂体制备 巨噬细胞摄取研究。这将使用荧光标记的IC载脂体和 表达GFP的巨噬细胞摄取和透射电子显微镜观察。

Project Summary/Abstract Non-tuberculous mycobacteria (NTM) are widespread pathogens found in the environment and cause progressive lung disease as well as skin and soft tissue, central nervous system and disseminated infections. NTM infections rates are rising globally and have emerged as important human pathogens globally. The specific pathogens responsible for these infections are part of one of two species, i) Mycobacterium abscessus (M. abscessus) complex (MABSC) and ii) Mycobacterium avium (M. avium) complex. The M. abscessus complex comprises the subspecies M. abscessus, M. massiliense and M. bolletii, has emerged as a significant global threat causing an increasing number of pulmonary infections among patients with structural lung disease such as chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis (CF). Of particular concern, patients with CF co-infected with an MABSC pathogen are often untreatable despite years of combination therapy resulting in 60-70% treatment failures. Therefore, novel anti-NTM agents with potent activity against MABSC strains that can shorten treatment time, decrease resistance rates with improved efficacy are strongly needed. We have discovered a novel series of indole-2-carboxamides (IC) that have potent minimum inhibitor concentration (MIC) values of 0.0039 - 8 µg/ml against various slow- and fast-growing NTM of clinical interest, including M. abscessus, M. massiliense, M. bolletii and M. chelonae. In addition, ICs are effective in vitro and in mice, demonstrating their safe and effective profile. However, ICs are lipophilic and are poorly water soluble resulting in poor oral absorption. To circumvent this poor pharmacokinetic property, we propose to design novel amino-acid based IC analogs and develop drug-loaded liposomes, which are a safe and effective option to formulate poorly soluble ICs. Novel analogs will be evaluated for antimycobacterial activity and active compounds will undergo a series of ADMETox assays to determine putative PK/PD action. IC- and IC-analog- loaded liposomes will be designed for inhalation administration and be engineered to be preferentially taken up by macrophages, where NTM pathogens reside in infected patients. This will be accomplished through synthesis of ICs, production of drug-loaded liposomes and characterization of drug release and macrophage uptake. Liposomes will be produced using thin film hydration method with subsequent IC-loading using passive trapping methods. The lamellarity of the liposomes will be monitored by 31P NMR and confirmed with cryo-TEM. Drug-release studies will be performed to ensure therapeutic IC concentrations are released (concentrations above MIC values). Finally, IC-loaded liposomes with optimized drug-release will be subjected to macrophage uptake studies. This will be performed using a fluorescent labeled IC loaded liposome and macrophages expressing GFP to visualize uptake and TEM.