Disrupting Biofilm Formation to Improve TB Drug Treatment

描述（由申请人提供）：结核分枝杆菌（Mtb）感染导致肺部和其他组织的慢性炎症，这是目前结核病（TB）药物难以治疗的。有效治愈活动性结核病患者的挑战是由于存在于干酪样坏死或空洞的肉芽肿中的耐药杆菌的持续存在。现有的治疗方案往往因毒副作用而复杂化，包括数月或数年的联合抗微生物药物，这被认为是有效根除耐药杆菌所必需的。我们提出了一种新的结核病治疗策略，使用一类小分子量的抗生物膜化合物与传统的结核病药物相结合，靶向耐药结核分枝杆菌。这一策略可以通过更快地根除持久性耐药杆菌来显著提高目前结核病药物治疗的疗效。我们的体外和体内数据表明，结核分枝杆菌形成细胞外的生物膜样微生物群落作为一种策略，以生存结核药物治疗。通过研究豚鼠TB模型，我们确定这些耐药杆菌在与溶解或坏死的白细胞相关时采用生物膜样存在模式。为了模拟这种独特的体内微环境，我们开发了一种新的体外试验，其中Mtb的细胞外生物膜样社区在来自裂解的人白细胞的附着基质上培养。与体内观察到的相似，我们的数据显示Mtb表达极端的体外药物耐受性，其被抗生物膜药物逆转，使杆菌再次对TB药物敏感。我们的数据表明，这些新型化合物分散或抑制由多种细菌形成的生物膜的形成，包括M。增加结核分枝杆菌对异烟肼的敏感性。该策略的有效性将使用我们的体外Mtb药物耐受性测定进一步测试，以显示1）抗生物膜 化合物在体外分散并抑制Mtb生物膜样群落的形成，和2）将抗生物膜化合物与TB药物组合有效地根除与宿主衍生的大分子相关的耐药性Mtb。然后，我们将确定在豚鼠Mtb感染模型中组合抗生物膜和TB药物是否比单独的TB或生物膜药物更有效地消除耐药性Mtb。在一系列机制研究中，我们将使用生物素化的抗生物膜化合物作为分子探针来鉴定耐药表型所特有的Mtb特异性毒力因子。通过确定耐药性Mtb特有的代谢途径的Mtb粘附素或蛋白质中间体的身份，将设计、合成并测试其他化合物的生物活性。这些高度创新的转化研究的成功完成将确立使用抗生物膜化合物作为辅助疗法与现有TB药物联合治疗慢性Mtb的可行性。我们还将确定抗生物膜化合物与结核病药物联合使用是否比单独使用结核病药物更有效地靶向耐药杆菌。最后，我们将揭示我们目前的抗生物膜化合物的作用机制和耐药结核分枝杆菌特有的其他可药用靶标的身份。

DESCRIPTION (provided by applicant): Infection with Mycobacterium tuberculosis (Mtb) results in chronic inflammation of the lung and other tissues, which is difficult to treat with current tuberculosis (TB) drugs. The challenge to effectively curing patients with active TB is due to the persistence of drug-tolerant bacilli sequestered in granulomas with caseous necrosis or cavitation. Existing treatment regimes that are often complicated by toxic side effects consist of months or years of combination antimicrobial drugs, which are deemed necessary to effectively eradicate drug-tolerant bacilli. We propose a novel TB treatment strategy, targeting drug-tolerant Mtb using a class of small molecular weight, anti-biofilm compounds to be combined with conventional TB drugs. This strategy could significantly improve the efficacy of current TB drug therapy by more rapidly eradicating, persistent, drug-tolerant bacilli. Our in vitr and in vivo data show that Mtb forms extracellular, biofilm-like microbial communities as a strategy to survive TB drug therapy. From studying the Guinea pig TB model we determined that these drug-tolerant bacilli adopt the biofilm-like mode of existence when associated with lysed or necrotic leukocytes. To mimic this unique in vivo microenvironment we developed a novel in vitro assay in which extracellular, biofilm-like communities of Mtb are cultured on an attachment matrix derived from lysed human leukocytes. Similar to what is seen in vivo, our data show that Mtb expresses extreme in vitro drug tolerance, which is reversed by anti-biofilm drugs, rendering bacilli again susceptible to TB drugs. Our data show that these novel compounds disperse or inhibit the formation of biofilms formed by a variety of bacteria including M. smegmatis and increases the susceptibility of Mtb to isoniazid. The effectiveness of this strategy will be further tested using our in vitro Mtb drug tolerance assay to show 1) anti-biofilm compounds disperse and inhibit the formation of Mtb biofilm-like communities in vitro and 2) that combining anti-biofilm compounds with TB drugs effectively eradicates drug-tolerant Mtb associated with host-derived macromolecules. We will then determine whether combining anti-biofilm and TB drugs in the Guinea pig Mtb infection model is more effective at eliminating drug- tolerant Mtb than TB or biofilm drugs alone. In a series of mechanistic studies, we will use biotinylated, anti- biofilm compounds as molecular probes to identify Mtb specific virulence factors unique to the drug-tolerant phenotype. By determining the identity of Mtb adhesins or protein intermediates of metabolic pathways unique to drug-tolerant Mtb, additional compounds will be designed, synthesized and tested for biological activity. Successful completion of these highly innovative, translational studies will establish the feasibility of using anti- biofilm compounds as adjunct therapy to treat chronic Mtb in conjunction with existing TB drugs. We will also determine if anti-biofilm compounds combined with TB drugs are more effective at targeting drug-tolerant bacilli compared to TB drugs alone. Finally, we will reveal the mechanisms of action of our current anti-biofilm compounds and the identity of other druggable targets unique to drug-tolerant Mtb.