Spectinomycin analogs for NTM infections

非结核分枝杆菌（NTM）是一种具有高度内在耐药性的新兴病原体。结核分枝杆菌是致病性最强的难治性NTM成员，感染这种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治疗在很高比例的病例中失败，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，但由于内在的耐药机制，在分枝杆菌中的疗效有限。对半合成的壮观霉素类似物的文库进行抗M.从该菌株中鉴定出一个独特的亚类该亚类的起始导联显示有效的抗M。因此，本发明的组合物具有良好的抗氧化活性，同时保持所需的药理学性质，即最小的细胞毒性和肝代谢、低蛋白结合和不存在线粒体蛋白合成抑制。这些先导化合物对多药耐药M.结核分枝杆菌临床分离株对其他NTM病原体有活性，最低程度地诱导WhiB 7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M中表现出稳健的疗效。小鼠感染模型。这些初步研究的结果表明，eAmSPC有潜力发展成为治疗M。其他NTM感染。该提案的主要目标是提高eAmSPC的效力和耐受性。这将通过迭代药物循环来实现，包括：（i）将对乙基侧链应用结构指导的优化策略以降低其亲脂性并在RpsE /30 S螺旋-34结合侧口袋中产生额外的结合相互作用。(ii)进一步的优化将通过分枝杆菌核糖体抑制、对一组非结核分枝杆菌（NTM）的最小抑菌浓度（MIC）活性和体外药代动力学研究来指导。最近开发的冷冻电镜方法在分枝杆菌核糖体将确认结合模式，并协助这些基于结构的药物设计工作。研究渗透性和外排的累积研究将有助于确定哪些结构修饰能够成功克服内在耐药机制。全基因组测序和RNAseq研究将确保化合物保持在目标上，并探索耐药性和毒力机制。(iii)将使用NTM感染的急性和慢性小鼠模型（重现NTM感染人肺的病理学），与标准治疗抗生素进行比较，确定新电极导线的体内药代动力学、安全性和疗效研究。将对晚期电极导线进行体外药理学安全性分析。在该提案中开发的化合物对M.将优先考虑包括那些对M有活动的人。禽流感病毒和其他非结核菌，以便产生针对这一重要病原体组的更广谱的治疗剂。

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.