Therapeutics for Drug-Resistant Bacteria: Pseudouridimycins

描述（由申请人提供）：我们发现了第一个选择性抑制细菌RNA聚合酶（RNAP）的核苷类似物抑制剂（NAI）：假尿嘧啶霉素（PUM）。PUM由链霉菌NAI 38640产生，包含与5 ′-氨基-假吡啶缀合的胍基化的N-羟基化Gly-Gln二肽。PUM在体外抑制细菌RNAP-但不抑制哺乳动物RNAP-，抑制培养物中的细菌生长，并在A组链球菌感染的小鼠模型中有效清除感染（ED 50 = 10 mg/kg）。该化合物对广谱的药物敏感性和药物抗性细菌病原体，包括药物敏感性、青霉素抗性、大环内酯抗性， 和多药耐药链球菌，药物敏感的、耐甲氧西林的和多药耐药的葡萄球菌，奈瑟氏菌属，嗜血杆菌属，该化合物与目前用于广谱抗菌治疗的RNAP抑制剂利福平不显示交叉耐药性，并且显示自发耐药频率<利福平的1/10。该化合物在与利福平共同给药时表现出附加的抗菌活性。 我们确定了PUM在RNAP上的结合位点（“i+1”NTP插入位点）和PUM抑制RNAP的机制（与UTP竞争“i+1”NTP插入位点）。结合位点和机制与RNAP抑制剂利福平的结合位点和机制没有重叠，这与不存在与利福平的交叉耐药性一致。 我们已经确定了与PUM复合的RNAP的晶体结构。晶体结构表明PUM的结构发生了特定的改变，预计将增加对广谱细菌RNAP的效力，利用细菌RNAP中不变的结构特征。 我们已经开发了PUM类似物的半合成和全合成的程序。 我们建议利用初步工作中获得的机制信息，结构信息和合成程序，以设计，合成和评估PUM类似物，其对广谱耐药和耐药细菌病原体具有更高的功效。将评价类似物的体外RNAP抑制、培养物中的抗菌活性、培养物中对哺乳动物细胞的细胞毒性、培养物中的抗性特性和物理特性。将在小动物感染模型中评估高前景的类似物的抗菌功效，并将评估最高前景的类似物的生物利用度，药代动力学，安全性和规模合成的能力。

DESCRIPTION (provided by applicant): We have discovered the first nucleoside-analog inhibitor (NAI) that selectively inhibits bacterial RNA polymerase (RNAP): pseudouridimycin (PUM). PUM is produced by Streptomyces sp. NAI38640 and comprises a guanidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 5'-amino-pseudoridine. PUM inhibits bacterial RNAP--but not mammalian RNAP--in vitro, inhibits bacterial growth in culture, and potently clears infection in a mouse model of Group A Streptococcus infection (ED50 = 10 mg/kg). The compound exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens, including drug-sensitive, penicillin-resistant, macrolide-resistant, and multi-drug-resistant Streptococci, drug-sensitive, methicillin-resistant, and multi-drug-resistant Staphylococci, Neisseria sp., Haemophilus sp., and Moraxella sp. The compound exhibits no cross-resistance with rifampin, the RNAP inhibitor in current use in broad-spectrum antibacterial therapy, and exhibits a spontaneous resistance frequency <1/10 that of rifampin. The compound exhibits additive antibacterial activity upon co-administration with rifampin. We have defined the binding site on RNAP for PUM (the "i+1" NTP insertion site) and the mechanism of inhibition of RNAP by PUM (competition with UTP for occupancy of the "i+1" NTP insertion site). The binding site and mechanism have no overlap with the binding site and mechanism of the RNAP inhibitor rifampin, consistent with the absence of cross-resistance with rifampin. We have determined a crystal structure of RNAP in complex with PUM. The crystal structure suggests specific alterations to the structure of PUM that are expected to increase potency against a broad spectrum of bacterial RNAP, exploiting structural features that are invariant in bacterial RNAP. We have developed procedures for semi-synthesis and total synthesis of PUM analogs. We propose to leverage the mechanistic information, structural information, and synthetic procedures obtained in preliminary work in order to design, synthesize, and evaluate PUM analogs having increased efficacy against a broad spectrum of drug-resistant and drug-resistant bacterial pathogens. Analogs will be evaluated for inhibition of RNAP in vitro, antibacterial activity in culture, cytotoxicity against mammalian cells in culture, resistance properties in culture, and physical properties. Analogs of high promise will be evaluated for antibacterial efficacy in small-animal models of infection, and analogs of highest promise will be evaluated for bioavailability, pharmacokinetics, safety, and ability to scale synthesis.