Optimization of rifamycins to overcome intrinsic resistance of nontuberculous mycobacteria to improve treatment of NTM lung disease

摘要 非结核分枝杆菌(NTM)是一种普遍存在的环境细菌，由快速和缓慢的细菌组成。 在患者中生长(RGM和SGM)条件致病菌并导致结核(TB)样肺部疾病 既有肺部疾病或免疫力受损。最常遇到的RGM和SGM 分别是脓肿分枝杆菌和禽分枝杆菌复合体(MAC)。其他临床上重要的NTM 包括偶然分枝杆菌和龟分枝杆菌(RGM)，以及堪萨斯分枝杆菌、遗传分枝杆菌、Xenopi分枝杆菌和猿猴分枝杆菌(SGM)。 这种治疗通常长达数年，主要包括重新调整用途和效果不佳的抗生素组合。 对于许多NTM疾病，没有可靠的治疗方案，死亡率很高，我们的首要目标是 优化利福霉素以克服内在耐药，提高NTM肺部疾病的治疗水平。 利福平(RIF)是结核病治疗的支柱药物，因其对专性病原体M。 结核(Mtb)，良好的药代动力学和良好的对疾病部位的渗透性。尽管RIF 被推荐用于治疗除猿猴外的所有sgm肺部疾病，其治疗效果尚未见报道。 除堪萨斯分枝杆菌病外，已经建立，与RIF对堪萨斯分枝杆菌和 结核分枝杆菌，但对所有其他非结核分枝杆菌的积极作用很差。利福霉素对大多数NTM不能达到可接受的疗效 由内在细菌耐药性引起的疾病，与其靶点的多态性或突变无关 RNA聚合酶的RpoB亚基。相反，我们已经表明，脓肿分枝杆菌经历了细菌内 代谢受利福霉素单加氧酶(S)(ROX)和腺苷二磷酸核糖苷酶(ARR)。通过系统基因组学 通过搜索，我们在所有主要的RGM和几个SGM中都发现了这些代谢酶。M.Kansassii，排好队 对利福平治疗有良好反应，ARR阴性。利福霉素糖基化酶和磷酸化酶， 在其他细菌中发现的，是一些非结核分枝杆菌内在耐药性的潜在额外候选者。 我们建议表征非结核分枝杆菌的种特异性利福霉素耐药组，并利用这一知识来 克服内源性耐药合理优化利福霉素类药物提高NTM肺治疗水平 疾病。在初步研究中使用耐ROX的利福布汀(RBT)作为化学起点，我们已经阻止了 ADP-核糖化，导致显著提高对脓肿分枝杆菌的效力，类似于RIF 针对Mtb(它不窝藏ROX或ARR)。我们将把这一方法扩展到适当的RGM和SGM 以抗药性发现为指导的物种。提供治疗多发性硬化症的临床前开发候选药物。 脓肿和其他ARR阳性的NTM肺部疾病，药物化学努力将集中在降低血浆 细胞色素P450诱导的蛋白质结合和消除药物与药物的相互作用，同时保持 效力和对肺部病变的良好穿透性。通过体外和小鼠模型的联合研究， 我们将确定最佳合作伙伴药物，以提供基于利福霉素的全口服杀菌药物组合，这些药物可以改善 提高治愈率，缩短疗程。

ABSTRACT Non-tuberculosis mycobacteria (NTM) are ubiquitous environmental bacteria comprising rapid- and slow- growing (RGM and SGM) opportunistic pathogens and causing tuberculosis (TB)-like lung disease in patients with pre-existing lung conditions or compromised immunity. The most frequently encountered RGM and SGM are Mycobacterium abscessus and the M. avium complex (MAC), respectively. Other clinically important NTM include M. fortuitum and M. chelonae (RGM), and M. kansasii, M. genavense, M. xenopi, and M. simiae (SGM). The often multiyear-long treatment consists of mostly repurposed and underperforming antibiotic combinations. For many NTM diseases, there is no reliable curative regimen and mortality is high Our overarching goal is to optimize rifamycins to overcome intrinsic resistance and improve treatment of NTM lung disease. Rifampicin (RIF) is the pillar of TB therapy owing to its exquisite potency against the obligate pathogen M. tuberculosis (Mtb), favorable pharmacokinetics and excellent penetration to the sites of disease. Although RIF is recommended for the treatment of all SGM pulmonary diseases but M. simiae, its therapeutic utility has not been established except for M. kansasii disease, in line with RIF being similarly potent against M. kansasii and Mtb but poorly active against all other NTMs. Rifamycins do not achieve acceptable efficacy against most NTM diseases due to intrinsic bacterial resistance not associated with polymorphisms or mutations in their target, the RpoB subunit of the RNA polymerase. Rather, we have shown that M. abscessus undergoes intrabacterial metabolism by rifamycin monooxygenase(s) (ROX) and ADP-ribosylase (Arr). Through systematic genomics searches, we have identified these metabolic enzymes in all major RGM and several SGM. M. kansassii, in line with its favorable response to rifampicin treatment, is Arr-negative. Rifamycin glycosylases and phosphorylases, discovered in other bacteria, are potential additional candidates contributing to intrinsic resistance in some NTM. We propose to characterize the species-specific rifamycin resistome of NTMs and exploit this knowledge to overcome intrinsic resistance and rationally optimize the rifamycin class to improve the treatment of NTM lung disease. Using ROX-resistant rifabutin (RBT) as chemical starting point in preliminary studies, we have blocked ADP-ribosylation, resulting in a dramatic potency improvement against M. abscessus, similar to that of RIF against Mtb (which does not harbor ROX or Arr). We will expand this approach to appropriate RGM and SGM species as guided by resistome findings. To deliver a preclinical development candidate for the treatment of M. abscessus and other Arr-positive NTM lung diseases, medicinal chemistry efforts will focus on reducing plasma protein binding and removing drug-drug interactions due to cytochrome P450 induction, while maintaining potency and favorable penetration into lung lesions. Through combination studies in vitro and in mouse models, we will identify best partner drugs to deliver all-oral bactericidal rifamycin-based combinations that can improve cure rates and shorten treatment duration.