Therapeutics for Drug-Resistant Bacteria: Myxopyronins

性状（由申请方提供）：Myxopyronin（Myx）是一种？-吡喃酮抗生素，通过与RNAP“开关区”相互作用抑制细菌RNA聚合酶（RNAP），RNAP“开关区”是介导RNAP结合并保留转录中的DNA模板所需的构象变化的结构元件。Myx不抑制真核RNAP I、RNAP II或RNAP III。Myx对培养物中的结核分枝杆菌、金黄色葡萄球菌、肺炎链球菌、粪肠球菌、阴沟肠杆菌和艰难梭菌具有强效抗菌活性。Myx与目前临床上用于治疗细菌感染的细菌RNAP抑制剂（利福霉素抗菌剂、利福平、利福喷丁和利福替尼）没有表现出交叉耐药性，并且与正在评估的其他细菌RNAP抑制剂没有或表现出最小的交叉耐药性，以用于未来临床上用于治疗细菌感染。在初步工作中，我们已经表明，Myx的功能是通过抑制细菌RNAP通过结合位点和机制，这是不同于利福霉素抗菌剂。我们已经确定了与Myx复合的细菌RNAP的晶体结构，并且我们已经构建了与Myx复合的病原体RNAP的同源模型。晶体结构和同源性模型表明Myx结构的改变，预期（i）特异性增加抗M的效力。结核RNAP，利用存在于M.结核RNAP，或（ii）通常增加针对广谱细菌RNAP的效力，利用广谱细菌RNAP中存在的结合位点界面水分子和其他结构特征。在进一步的初步工作中，我们优化了Myx和Myx类似物的全合成程序，开发了制备重组病原体RNAP的程序，并开发了病原体RNAP的荧光和放射化学测定程序。我们建议利用初步工作中开发的机制和结构信息、合成程序和测定程序，以设计、合成和评估：（i）对多药耐药和广泛耐药M具有增加的功效的Myx类似物。结核病，和（ii）对广谱耐药病原体具有增加的功效的Myx类似物。将评价类似物的体外RNAP抑制、培养物中的抗菌活性和培养物中对哺乳动物细胞的细胞毒性。将在小动物感染模型中评估高前景的类似物的抗菌活性，并将评估最高前景的类似物的生物利用度，药代动力学，毒性和规模合成的能力。主要目标病原体包括：M.结核病H37 Rv和MDR/XDR、金黄色葡萄球菌MSSA和MRSA、粪肠球菌VSE和VRE、肺炎链球菌、阴沟肠杆菌和艰难梭菌。耐药性细菌感染是一个主要的和日益增长的威胁。预计拟议的工作将提供两类新的候选药物：（1）有效对抗多重耐药和广泛耐药结核病的抗菌剂，以及（2）有效对抗广谱耐药细菌病原体的抗菌剂，包括公共卫生相关细菌病原体和生物防御相关细菌病原体。

DESCRIPTION (provided by applicant): Myxopyronin (Myx) is an ?-pyrone antibiotic that inhibits bacterial RNA polymerase (RNAP) through interactions with the RNAP "switch region," a structural element that mediates conformational changes required for RNAP to bind and retain the DNA template in transcription. Myx does not inhibit eukaryotic RNAP I, RNAP II, or RNAP III. Myx exhibits potent antibacterial activity against Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Enterobacter cloacae, and Clostridium difficile in culture. Myx exhibits no cross-resistance with the inhibitors of bacterial RNAP in current clinical use in therapy of bacterial infection (the rifamycin antibacterial agents, rifampin, rifapentine, and rifabutin), and exhibits no or minimal cross-resistance with other inhibitors of bacterial RNAP under evaluation for future clinical use in therapy of bacterial infection. In preliminary work, we have shown that Myx functions by inhibiting bacterial RNAP through a binding site and mechanism that are different from those of rifamycin antibacterial agents. We have determined a crystal structure of a bacterial RNAP in complex with Myx, and we have constructed homology models of pathogen RNAP in complex with Myx. The crystal structure and homology models suggest alterations to the structure of Myx that are expected (i) specifically to increase potency against M. tuberculosis RNAP, exploiting a binding-site cysteine residue present in M. tuberculosis RNAP, or (ii) generally to increase potency against a broad spectrum of bacterial RNAP, exploiting a binding-site interfacial water molecule, and other structural features, present in a broad spectrum of bacterial RNAP. In further preliminary work, we have optimized procedures for total synthesis of Myx and Myx analogs, developed procedures for preparation of recombinant pathogen RNAP, and developed procedures for fluorescent and radiochemical assays of pathogen RNAP. We propose to leverage the mechanistic and structural information, synthetic procedures, and assay procedures developed in preliminary work in order to design, synthesize, and evaluate: (i) Myx analogs with increased efficacy against multidrug-resistant and extensively-drug-resistant M. tuberculosis, and (ii) Myx analogs with increased efficacy against a broad spectrum of drug-resistant pathogens. Analogs will be evaluated for inhibition of RNAP in vitro, for antibacterial activity in culture, and for cytotoxicity against mammalian cells in culture. Analogs of high promise will be evaluated for antibacterial activity in small-animal models of infection, and analogs of highest promise will be evaluated for bioavailability, pharmacokinetics, toxicity, and ability to scale synthesis. Primary target pathogens include: M. tuberculosis H37Rv and MDR/XDR, Staphylococcus aureus MSSA and MRSA, Enterococcus faecalis VSE and VRE, Streptococcus pneumoniae, Enterobacter cloacae, and Clostridium difficile. Drug-resistant bacterial infections are a major and growing threat. The proposed work is expected to provide two classes of new drug candidates: (1) antibacterial agents effective against multi-drug-resistant and extensively-drug-resistant tuberculosis, and (2) antibacterial agents effective against a broad spectrum of drug-resistant bacterial pathogens, including both public-health-relevant bacterial pathogens and biodefense-relevant bacterial pathogens.