Developing novel pyrazolidinone antibiotics targeting PBP3 to overcome resistance mechanisms

青霉素结合蛋白(PBPS)是β-内酰胺类药物的靶点，但对β-内酰胺类药物耐药 抗生素，如碳青霉烯类和头孢菌素类，已导致全球健康问题。有一种 多种耐药机制，其中降解β-内酰胺酶的β-内酰胺酶是主要原因之一。 我们的目标是通过研究和研究克服经常与β-内酰胺类药物相关的耐药机制 开发一种不同类型的PBP抑制剂--吡唑烷酮。吡唑烷酮类化合物YU253434和 YU253911含有铁载体部分，以促进铁介导的摄取。我们发现这两个人 吡唑烷酮类化合物不能被A、C和D类β-内酰胺酶水解，只能缓慢水解 由(B类)金属β-内酰胺酶。YU253434和YU253911也比氨曲南有利， 头孢他啶、美罗培南、头孢他啶/他唑巴坦、头孢他啶/阿维巴坦 对铜绿假单胞菌、肺炎克雷伯氏菌、大肠杆菌和 鲍曼不动杆菌(四种都是ESKAPE病原体)。YU253911还显著降低了菌落总数。 多药耐药铜绿假单胞菌株小鼠大腿感染模型中的形成单位。吡唑烷酮类药物靶标 和抑制PBP3，我们从结晶学的角度描述了它们与铜绿假单胞菌PBP3的结合方式。 总体而言，我们发现这些吡唑烷酮具有几个有利的属性，但仍有进一步的改进 在克服已知的PBP3 F533L抗性所需的PBP3 IC50、摄取和能力方面 突变。我们在以下两个具体目标中提出了这些改进措施。 具体目的1.通过针对R2的基于结构的修饰来提高吡唑烷酮的亲和力 一群人。基于晶体结构，我们假设疏水取代基添加到 铁载体连接体中的吡唑烷酮类化合物将与疏水桥残基F533和V333相互作用 从而提高亲和力。此外，这些疏水相互作用旨在 中和F533L抗性突变。我们将对这些化合物进行微生物学测试 经过充分研究的肺炎克雷伯菌、鲍曼不动杆菌、大肠杆菌和铜绿假单胞菌对PBP的抑制作用 WT和F533L铜绿假单胞菌PbP3，并从结晶学、生物物理、生物化学等方面探讨了它们的结合方式。 并使用分子动力学模拟。 具体目的2.通过添加提高铁非依赖性和铁介导性的吡唑烷酮的摄取 铁载体连接体的含胺部分(目标2a)，并通过将铁载体与 与羟基相邻的吸电子-氯基团以提高铁的亲和力(目标2b)。 这项高风险、高回报的提案旨在开发一种更有效的非β-内酰胺类药物 这可能导致一种新的治疗策略来对抗抗生素耐药性。

Penicillin-binding proteins (PBPs) are a proven β-lactam drug target, yet resistance to β-lactam antibiotics, such as carbapenems and cephalosporins, has resulted in a global health problem. There are a number of resistance mechanisms of which β-lactam degrading β-lactamases is one of the main culprits. Our goal is to overcome the resistance mechanisms often associated with β-lactams by studying and developing a different type of PBP inhibitor, the pyrazolidinone. The pyrazolidinones YU253434 and YU253911 contain a siderophore moiety to facilitate iron-mediated uptake. We have found that these two pyrazolidinones cannot be hydrolyzed by Classes A, C, and D β-lactamases and are only slowly hydrolyzed by (Class B) metallo β-lactamases. YU253434 and YU253911 also compared favorably to aztreonam, ceftazidime, meropenem, ceftolozane/tazobactam, and ceftazidime/avibactam when microbiologically tested against panels of Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii (all four are ESKAPE pathogens). YU253911 also significantly lowered colony- forming units in a mouse thigh-infection model with an MDR P. aeruginosa strain. The pyrazolidinones target and inhibit PBP3, and we have delineated their binding modes to P. aeruginosa PBP3 crystallographically. Overall, we found that these pyrazolidinones have several favorable attributes, yet further improvements are needed in terms of PBP3 IC50, uptake, and ability to overcome the known PBP3 F533L resistance mutation. We propose these improvements in the following two specific Aims. Specific Aim 1. To improve the pyrazolidinone affinity via structure-based modifications targeting the R2 group. Based on the crystal structure, we hypothesize that hydrophobic substituents added to pyrazolidinones in the siderophore-linker will interact with the hydrophobic bridge residues F533 and V333 in PBP3 and thereby improve affinity. Additionally, these hydrophobic interactions are designed to counteract the F533L resistance mutation. We will test the compounds microbiologically against panels of well-studied K. pneumoniae, A. baumannii, Escherichia coli, and P. aeruginosa, measure PBP inhibition both wt and F533L P. aeruginosa PBP3, and probe their binding mode crystallographically, biophysically, and using molecular dynamics simulations. Specific Aim 2. To improve the iron–independent and iron-mediated uptake of pyrazolidinones via adding an amine-containing moiety to the siderophore-linker (Aim 2a) and by incorporating a siderophore with an electron-withdrawing –Cl group adjacent to the hydroxyl groups to improve iron affinity (Aim 2b). This high-risk, high-reward proposal aims to develop a more potent non-β-lactam PBP-targeting pyrazolidinone that could lead to a novel therapeutic strategy to combat antibiotic resistance.