Overcoming Pyrazinamide Resistance with Pyrazinoate-Cephalosporin Conjugates

摘要 结核分枝杆菌(Mtb)是结核病(TB)的主要病原体，感染了超过三分之一的 目前已成为单一病原体导致传染病死亡的主要原因。吡津酰胺 PZA是一种一线杀菌抗结核药物，有望成为未来抗结核药物中不可替代的组成部分 结核病治疗方案。以前的研究表明，PZA是一种前药物，它被转化为 结核分枝杆菌酰胺酶PncA活性形式吡嗪酸(POA)及其功能突变丧失 在pncA中，PZA抗性占绝大多数。对PZA的耐药性反过来又与 显著更高的治疗失败和复发率。尝试在人类体内直接给药POA和 小鼠没有成功，因为POA在感染时不能在结核分枝杆菌中达到足够的浓度 地点。使用简单POA酯的前药方法一直无法克服固有的药物 与常规前药一样，POA的处置能力被血清酯酶迅速降解。在……里面 初步数据，我们已经开发了一种通过与β-内酰胺偶联来治疗POA的新的前药策略 独特的分枝杆菌β-内酰胺酶Blac促进POA的选择性释放而不是 血清酯酶。这一策略通过绕过pncA介导的激活和 使BLAC成为条件必需的，因为BLAC失去了功能突变，恢复了Mtb对 β-内酰胺的促销活动。因此，我们假设自发性发育的遗传障碍 对我们的POA-β-内酰胺结合物的抵抗力将很高。这个应用程序的目标是合成和 评价POA的β-内酰胺前药，这些药物是口服生物利用度的，并在Mtb-Mtb内选择性释放POA。 感染的巨噬细胞。我们将通过追求两个具体的目标来实现此应用程序的总体目标 目标。本研究的目的是合成β-内酰胺类POA前体药物，通过mtb选择性激活释放。 POA而不是共生微生物区系。前药将进行生物化学和微生物学表征 一组β-内酰胺酶和微生物。在目标2中，我们将测量前药物在血清和 刺激胃液和Caco-2细胞通透性。接下来我们将确定完整的药代动力学 肠道微生物群释放POA的参数和程度。最后，我们计划评估一种化合物 在小鼠结核病感染模型中。

SUMMARY Mycobacterium tuberculosis (Mtb), the principal etiological agent of tuberculosis (TB), infects over one-third of humanity and is now the leading cause of infectious disease mortality by a single pathogen. Pyrazinamide (PZA) is a first-line sterilizing anti-tubercular drug that is anticipated to be an irreplaceable component of future TB treatment regimens. Previous studies have demonstrated that PZA is a pro-drug which is converted to the active form of pyrazinoic acid (POA) by the M. tuberculosis amidase PncA, and that loss of function mutations in pncA account for the vast majority of PZA resistance. Resistance to PZA in turn is associated with significantly higher treatment failures and relapse rates. Attempts to directly administer POA in humans and mice have been unsuccessful because POA cannot attain sufficient concentrations within Mtb at the infection site. Prodrug approaches employing simple POA esters have been unable to overcome the intrinsic drug disposition liabilities of POA as conventional prodrugs are rapidly hydrolyzed by serum esterases. In preliminary data, we have developed a novel prodrug strategy for POA through conjugation to a β-lactam promoiety leading to selective release of POA by the unique mycobacterial beta-lactamase BlaC and not by serum esterases. This strategy overcomes PZA-resistance by circumventing PncA-mediated activation and renders blaC conditionally essential since loss of function mutations to blaC, restores susceptibility of Mtb to the β-lactam promoiety. Consequently, we hypothesize the genetic barrier for development of spontaneous resistance to our POA-β-lactam conjugates will be high. The objectives of this application are to synthesize and evaluate β-lactam prodrugs of POA, which are orally bioavailable and selectively release POA within Mtb- infected macrophages. We will accomplish the overall objectives of this application by pursuing two specific aims. In aim, we will synthesize β-lactam prodrugs of POA, which are selectively activated by Mtb to release POA and not by commensal microbiota. The prodrugs will be biochemically and microbiologically characterized with a panel of β-lactamases and microorganisms. In aim 2, we will measure prodrug stability in serum and stimulated gastric fluid and permeability in Caco-2 cells. We will next determine complete pharmacokinetic parameters and the extent of POA release by the gut microbiome. Finally, we plan to evaluate one compound in a murine TB infection model.