Spectinomycin analogs for NTM infections

非结核分枝杆菌(NTM)是一种新型致病菌，具有较高的内在耐药性。脓肿分枝杆菌是最具致病性的难治性NTM成员，感染这种病原体与临床预后特别差有关。阿米卡星和克拉霉素的标准护理治疗在很高比例的病例中失败，因此显然需要新的治疗方案。为了应对这一挑战，我们研究了壮观霉素的合成修饰，壮观霉素是一种氨基环素抗生素，它对细菌蛋白质合成具有很强的抑制作用，但由于内在耐药机制，对分枝杆菌的疗效有限。对半合成的壮观霉素类似物的抗脓肿活性进行了分析，从中鉴定出一个独特的乙烯连接氨甲基壮观霉素结构亚类(EAmSPC)。这一亚类的最初线索显示出很强的抗M。在保持最低细胞毒性和肝脏代谢、低蛋白质结合力和没有线粒体蛋白质合成抑制等所需药理特性的同时，还可抑制脓肿的活性。这些先导化合物对多重耐药脓肿分支杆菌临床分离株具有良好的活性，对其他NTM病原体具有活性，最小限度地诱导WhiB7核糖体应激反应途径，不是分枝杆菌氨基糖苷类修饰酶的底物，在脓肿分支杆菌小鼠感染模型中显示出强大的疗效。这些初步研究的结果表明，eAmSPC有可能被开发成治疗脓肿支原体和其他NTM感染的药物。这项建议的主要目标是提高eAmSPC的效力和耐受性。这将通过迭代药物循环实现，包括：(I)将结构导向的优化策略应用于乙基侧链，以降低其亲油性，并在RpsE/30S螺旋-34结合侧袋中产生额外的结合作用。(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发展的冷冻-EM方法将确认结合模式，并有助于这些基于结构的药物设计工作。研究渗透性和外流的累积研究将有助于确定哪些结构修饰在克服内在抗性机制方面是成功的。全基因组测序和RNAseq研究将确保化合物保持在靶标上，并探索耐药性和毒力机制。(Iii)将利用NTM感染的急性和慢性小鼠模型(概括了NTM感染的人类肺部的病理)，与护理抗生素的标准相比较，确定对新出现的铅的体内药代动力学、安全性和有效性研究。为了体外药理学安全性，将对晚期导联进行分析。在这项建议中开发的针对脓肿分枝杆菌的化合物将优先包括那些对禽类分枝杆菌和其他非结核分枝杆菌具有活性的化合物，以便产生针对这一重要病原体组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.