Target based discovery of next generation pyrazinamide

结核病(TB)是艾滋病患者的主要死亡原因。在免疫能力强的人群中，药物 治疗和免疫联手战胜结核分枝杆菌(Mtb)。在HIV阳性的人中 另一方面，化疗必须完全消毒所有感染部位。有一位急诊医生 需要开发更有效的“消毒”药物。 将吡嗪酰胺(PZA)纳入结核病方案使治疗时间减少到6个月，而 保持较低的复发率。PZA显著杀菌作用的药理学基础 考虑到该药物的体外效力较差(MIC=30-100微克/毫升)，该药物仍不清楚。在活动性兔模型中 TB是一种动物模型，它概括了在人类结核病中观察到的各种肺部病变类型，我们展示了 PZA不仅能穿透干酪性坏死性病变，还能对它们进行消毒。在使用干酪的体外试验中 在感染的兔子身上，我们发现PZA可以杀死不生长的、耐药的结核分枝杆菌。这些发现提供了一个 解释临床观察的PZA的治疗缩短效应：药物达到难以渗透 结核病损害和杀死顽固的“顽固不化”结核分枝杆菌。然而，与PZA适度的体外效力一致， 病变灭菌开始缓慢，体外杀灭结核分枝杆菌所需浓度很高。基于 这些发现，合乎逻辑的前进道路是提高PZA的效力，同时保持其独特的病变 渗透性和杀菌性能。为了实现PZA的合理优化，我们确定了天冬氨酸 脱羧酶PanD，辅酶A生物合成所必需的，作为第一个基因，生化和 PZA的生物物理验证靶标。与PZA全细胞活性差一致，药物的亲和力(更多 具体地说，其对PanD的生物活性成分吡津酸(POA)在微米范围内，证实了 进步。有趣的是，作用机制研究揭示了一种新的抗菌靶向机制。 因此，POA与PanD的结合似乎引发了降解，而不是抑制PanD的催化活性 酪蛋白水解酶ClpC1. 在这里，我们建议以我们的发现为基础，并1.充分描述新的靶上机制，通过它 药物诱导其靶标降解，2.体外药理‘灭菌’模型的建立和基于PanD的 用于发现具有改进的效力和杀菌活性的新的PZA类似物的检测，以及3.表征 抗PZA突变体Mtb与野生型Mtb在结核病兔体内的皮损特异性生长和复制状态 有无PZA治疗的模型。 总而言之，我们已经确定了与缩短治疗活动有关的关键药理特性。 PZA在临床环境中的应用，以及该药物的分子靶点，我们提出了一种从一流到一流的 利用这些发现并合理地设计具有改进灭菌功能的“下一代”PZA 活动。

Tuberculosis (TB) is a main cause of death for people living with AIDS. In immune-competent populations, drug therapy and immunity join forces to win the fight against Mycobacterium tuberculosis (Mtb). In HIV-positive individuals on the other hand, chemotherapy must fully sterilize all infection sites. There is an urgent medical need to develop more potent `sterilizing' drugs. The inclusion of pyrazinamide (PZA) into the TB regimen allowed reduction of treatment time to six months, while maintaining low relapse rates. The pharmacological basis for PZA's remarkable sterilizing activity in patients remains obscure, considering the drug's poor in vitro potency (MIC = 30-100 µg/mL). In the rabbit model of active TB, an animal model that recapitulates the various lung lesion types observed in human TB disease, we showed that PZA not only penetrates caseous necrotic lesions but also sterilizes them. In an ex vivo assay using caseum from infected rabbits, we showed that PZA kills non-growing, drug tolerant Mtb. These findings provide an explanation for the clinically observed treatment shortening effect of PZA: the drug reaches difficult-to-penetrate TB lesions and kills recalcitrant `persister' Mtb. However, consistent with the modest in vitro potency of PZA, onset of lesion sterilization is slow and concentrations required to kill Mtb in ex vivo caseum are high. Based on these findings, the logical way forward is to improve the potency of PZA while maintaining its unique lesion penetration and sterilization properties. To enable the rational optimization of PZA, we identified aspartate decarboxylase PanD, required for coenzymeA biosynthesis, as a the first genetically, biochemically and biophysically validated target of PZA. Consistent with poor whole cell activities of PZA, affinity of the drug (more specifically its bioactive component pyrazinoic acid, POA) for PanD was in the µM range, confirming room for improvement. Interestingly, mechanism of action studies revealed a novel antibacterial on-target mechanism whereby, rather than inhibiting PanD's catalytic activity, binding of POA to PanD appears to trigger degradation of the protein by the caseinolytic protease ClpC1. Here, we propose to build on our discoveries and 1. fully characterize the novel on-target mechanism by which the drug induces degradation of its target, 2. exploit in vitro pharmacological `sterilizing' models and PanD-based assays for the discovery of novel PZA analogs with improved potency and sterilizing activity, and 3. characterize lesion specific growth and replication status of PZA resistant panD mutant Mtb vs. wild type Mtb in the TB rabbit model with and without PZA treatment. In summary, we have identified key pharmacological properties responsible for the treatment shortening activity of PZA in clinical settings, as well as the molecular target of the drug, and we propose a `first-in-class to best-in- class' program to exploit these findings and rationally design the `next generation' PZA with improved sterilizing activity.