Spectinomycin analogs for NTM infections

非结核分枝杆菌（NTM）是一种具有高内在耐药性的新兴病原体。脓肿分枝杆菌是最具致病性的难治性NTM成员，感染该病原体与特别差的临床结果相关。阿米卡星和克拉霉素的标准护理治疗在很大比例的病例中失败，因此显然需要新的治疗选择。为了应对这一挑战，我们研究了大观霉素的合成修饰，大观霉素是一种氨基环醇抗生素，具有有效的细菌蛋白质合成抑制作用，但由于固有的耐药机制，对分枝杆菌的疗效有限。对半合成大观霉素类似物库进行了对脓疡分枝杆菌的活性分析，从中鉴定出乙烯连接氨基甲基大观霉素（eAmSPC）的独特结构亚类。该亚类的初始引线显示出有效的抗- m。脓肿活性，同时保持所需的药理特性，最小的细胞毒性和肝脏代谢，低蛋白结合，没有线粒体蛋白合成抑制。这些导联物对多药耐药脓肿支原体临床分离株有良好的活性，对其他NTM病原体有活性，最低限度地诱导WhiB7核糖体应激反应途径，不是分枝杆菌氨基糖苷修饰酶的底物，并在脓肿支原体小鼠感染模型中显示出强大的疗效。这些初步研究结果表明，eAmSPCs具有开发用于治疗脓肿支原体和其他NTM感染的潜力。本提案的主要目标是提高eamspc的效力和耐受性。这将通过一个迭代的药物周期来实现，包括：(i)在乙基侧链上应用结构导向的优化策略，以降低其亲脂性，并在RpsE / 30S螺旋-34结合侧袋中产生额外的结合相互作用。（ii）进一步的优化将以分枝杆菌核糖体抑制、对一组非结核分枝杆菌（NTM）的最低抑制浓度（MIC）活性和体外药代动力学研究为指导。最近在分枝杆菌核糖体中开发的冷冻电镜方法将证实这种结合模式，并有助于这些基于结构的药物设计工作。研究渗透性和外排的积累研究将有助于确定哪些结构修改是成功克服内在阻力机制的。全基因组测序和RNAseq研究将确保化合物保持在靶标上，并探索耐药和毒力机制。（iii）将使用急性和慢性NTM感染小鼠模型，重现NTM感染人类肺部的病理，确定新出现的铅的体内药代动力学、安全性和有效性研究，并与标准护理抗生素进行比较。后期的线索将被描述为体外药理学安全性。在本研究中开发的抗脓疡分枝杆菌的化合物将优先包括那些具有抗鸟分枝杆菌和其他ntm活性的化合物，以便产生针对这一重要病原体的更广泛的治疗方法。

Non-tuberculous mycobacteria (NTM) are emerging pathogens with high intrinsic drug resistance. Mycobacterium abscessus is the most pathogenic refractory NTM member, and infections with this pathogen are associated with especially poor clinical outcomes. The standard of care therapy of amikacin and clarithromycin fails in a high proportion of cases, and thus there is a clear need for new therapeutic options. To approach this challenge, we investigated the synthetic modification of spectinomycin, an aminocyclitol antibiotic that exhibits potent bacterial protein synthesis inhibition but has limited efficacy in mycobacteria due to intrinsic resistance mechanisms. A library of semi-synthetic spectinomycin analogs was profiled for activity against M. abscessus, from which a distinct structural subclass of ethylene linked aminomethyl spectinomycins (eAmSPC) was identified. Initial leads of this subclass display potent anti-M. abscessus activity, while maintaining the desired pharmacological properties of minimal cytotoxicity and hepatic metabolism, low protein binding, and absence of mitochondrial protein synthesis inhibition. These leads have favorable activity against multi-drug resistant M. abscessus clinical isolates, are active against other NTM pathogens, minimally induce the WhiB7 ribosomal stress response pathway, are not substrates for mycobacterial aminoglycoside modifying enzymes and demonstrate robust efficacy in M. abscessus mouse infection models. The results of these preliminary studies suggest that eAmSPCs have the potential to be developed into treatments for M. abscessus and other NTM infections. The key goals of this proposal are to increase the potency and tolerability of the eAmSPCs. This will be achieved through an iterative drug cycle to include: (i) A structure-guided optimization strategy will be applied to the ethyl side chain to reduce its lipophilicity and generate extra binding interactions in the RpsE / 30S helix-34 binding side pocket. (ii) Further rounds of optimization will be guided by mycobacterial ribosomal inhibition, minimum inhibitory concentration (MIC) activity against a panel of non-tuberculous mycobacteria (NTM), and in vitro pharmacokinetic studies. Recently developed Cryo-EM methods in mycobacterial ribosomes will confirm the binding mode and assist in these structure-based drug design efforts. Accumulation studies to investigate permeability and efflux will help define which structural modifications are successful in overcoming intrinsic resistance mechanisms. Whole- genome sequencing and RNAseq studies will ensure compounds remain on target and explore drug resistance and virulence mechanisms. (iii) In vivo pharmacokinetics, safety, and efficacy studies on emerging leads will be determined, in comparison to the standard of care antibiotics, using acute and chronic mouse models of NTM infection that recapitulate the pathology of NTM infected human lung. Late leads will be profiled for in vitro pharmacology safety. Compounds developed in this proposal against M. abscessus will be prioritized to include those with activity against M. avium and other NTMs in order to generate therapeutics with a wider spectrum against this important group of pathogens.