Therapeutics for Drug-Resistant Bacteria: Pseudouridimycins

描述（由申请人提供）：我们发现了第一种选择性抑制细菌RNA聚合酶（RNAP）的核苷类似物抑制剂（NAI）：假尿嘧啶（PUM）。PUM是由链霉菌（Streptomyces sp. NAI38640）生产的，它含有一种胍基化、n -羟基化的Gly-Gln二肽，与5'-氨基假嘌呤偶联。PUM在体外抑制细菌RNAP（但不抑制哺乳动物RNAP），在培养中抑制细菌生长，并在a组链球菌感染小鼠模型中有效清除感染（ED50 = 10 mg/kg）。该化合物对广谱的药物敏感和耐药细菌病原体具有抗菌活性，包括药物敏感、青霉素耐药、大环内酯耐药、

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.