Therapeutics for Drug-Resistant Bacteria: Myxopyronins

描述（由申请人提供）：Myxopyronin （Myx）是一种？-吡酮类抗生素，通过与RNAP“开关区”相互作用抑制细菌RNA聚合酶（RNAP）， RNAP“开关区”是一种结构元件，介导RNAP在转录中结合和保留DNA模板所需的构象变化。Myx不抑制真核RNAP I、RNAP II或RNAP III。在培养中，Myx对结核分枝杆菌、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与目前临床用于治疗细菌感染的细菌RNAP抑制剂（利福霉素、利福平、利福喷丁和利福布汀）没有交叉耐药，并且与其他正在评估的用于治疗细菌感染的细菌RNAP抑制剂没有或只有很小的交叉耐药。在前期工作中，我们已经证明Myx通过一个不同于利福霉素抗菌剂的结合位点和机制抑制细菌RNAP。我们确定了细菌RNAP与Myx复合物的晶体结构，并构建了病原体RNAP与Myx复合物的同源模型。晶体结构和同源性模型表明，Myx结构的改变有望(i)特异性地提高对结核分枝杆菌RNAP的效力，利用结核分枝杆菌RNAP中存在的结合位点半胱氨酸残基，或（ii）一般地提高对广谱细菌RNAP的效力，利用结合位点界面水分子和其他结构特征，存在于广谱细菌RNAP中。在进一步的前期工作中，我们优化了Myx和Myx类似物的全合成流程，开发了重组病原体RNAP的制备流程，并开发了病原体RNAP的荧光和放射化学检测流程。我们建议利用在初步工作中开发的机制和结构信息、合成程序和分析程序来设计、合成和评估：(i)对多药耐药和广泛耐药结核分枝杆菌具有更高疗效的Myx类似物，以及（ii）对广谱耐药病原体具有更高疗效的Myx类似物。研究人员将评估类似物在体外对RNAP的抑制作用、培养物的抗菌活性以及对哺乳动物细胞的细胞毒性。高希望的类似物将在小动物感染模型中进行抗菌活性评估，而最有希望的类似物将在生物利用度、药代动力学、毒性和大规模合成能力方面进行评估。主要目标病原体包括：结核分枝杆菌H37Rv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。耐药细菌感染是一个日益严重的重大威胁。拟议的工作预计将提供两类新的候选药物：(1)对多重耐药和广泛耐药结核病有效的抗菌药物，以及(2)对广谱耐药细菌病原体有效的抗菌药物，包括与公共卫生相关的细菌病原体和与生物防御相关的细菌病原体。

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.