Tofacitinib Analogs as Oxacillin Potentiators against MRSA

项目摘要 耐甲氧西林金黄色葡萄球菌（MRSA）感染是最常见的 正在发生的和危险的抗药性公共卫生威胁。对所有一线MRSA的临床耐药性 已观察到抗生素（即万古霉素、利奈唑胺、达托霉素和头孢洛林），进一步加重 这一威胁，并强调迫切需要新的战略，以克服耐药性在S。金黄色。在 近年来，人们对靶向信号转导系统的开发越来越感兴趣， 抗微生物剂和抗毒性剂。靶向细菌信号转导是有吸引力的，因为这些 系统对于原核生物中的许多细胞过程是必不可少的，并且信号转导途径已经被 在真核系统中进行了广泛的研究，如化学文库的可用性所证明的， 具有激酶通道样性质的小分子探针。考虑到这一战略，我们筛选了一个重点 一组激酶抑制剂，并确定托法替尼，一种抗风湿Janus激酶抑制剂，作为一种潜在的 发现抗MRSA的β-内酰胺增效剂的化学起点。几种吡咯并吡啶-4- 合成了托法替尼的胺（P4 A）类似物，这导致发现了具有托法替尼的有效类似物。 使苯唑西林（OXA;一种β-内酰胺抗生素）对MRSA重新敏感的能力。初步下拉蛋白质组学 实验已经确定PurM、SrrB和PknB为假定的靶点， 通过基因敲除实验。因此，本提案的主要科学目标是确认P4 A 使用生物物理和生物化学技术参与推定的靶点，并建立 托法替尼类似物作为抗病毒剂和OXA增效剂的治疗潜力 耐甲氧西林金黄色葡萄球菌在目标1中，我将评估P4 A先导化合物与推定靶点的结合，证明抑制 色葡萄金黄色葡萄球菌激酶活性，并确定对哺乳动物激酶的潜在脱靶效应的程度。 将通过一种新的靶点结合试验， 其将用生化磷酸化和ATP/ADP转化测定来补充。在目标2中，我 将通过抗生物膜研究、药理学ADME和药物代谢试验评估P4 A化合物的临床潜力。 在小鼠模型中的抗MRSA的体内功效。研究计划将加强我的训练， 目标识别和验证、生物物理分析和抗感染微生物学领域， 我的共同导师理查德·李博士和杰森·罗施博士的专业知识，以及 圣裘德

Project Summary Methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) infections are among the most frequently occurring and dangerous antibiotic-resistant public health threat. Clinical resistance to all front-line MRSA antibiotics (i.e. vancomycin, linezolid, daptomycin, and ceftaroline) has been observed, further exacerbating this threat and highlighting the urgent need for new strategies to overcome drug resistance in S. aureus. In recent years, there has been increased interest in targeting signal transduction systems for development as antimicrobials and anti-virulence agents. Targeting bacterial signal transduction is attractive because these systems are essential for many cellular processes in prokaryotes, and signal transduction pathways have been extensively studied in eukaryotic systems, as evidenced by the availability of chemical libraries containing small molecule probes with kinase inhibitor-like properties. With this strategy in mind, we screened a focused set of kinase inhibitors and identified tofacitinib, an antirheumatic Janus kinase inhibitor, as a potential chemistry starting point for the discovery of beta-lactam potentiators against MRSA. Several pyrrolopyridin-4- amine (P4A) analogs of tofacitinib were synthesized, which led to the discovery of potent analogs with the ability to re-sensitize oxacillin (OXA; a beta-lactam antibiotic) against MRSA. Preliminary pull-down proteomic experiments have identified PurM, SrrB, and PknB as putative targets, studies that have been complemented by gene knockout experiments. Therefore, the main scientific objective of this proposal is to confirm P4A engagement of putative targets using biophysical and biochemical techniques and to establish the therapeutic potential of tofacitinib analogs as anti-virulence agents and OXA potentiators against MRSA. In Aim 1, I will evaluate the binding of lead P4A compounds to putative targets, demonstrate inhibition of S. aureus kinase activity and determine the extent of potential off-target effects on mammalian kinases. Biophysical assays will be developed against the putative targets through a novel target engagement assay, which will be complemented with biochemical phosphorylation and ATP/ADP conversion assays. In Aim 2, I will evaluate the clinical potential of the P4A compounds through anti-biofilm studies, pharmacological ADME profiling, and in vivo efficacy against MRSA in a mouse model. The research plan will enhance my training in areas of target identification and validation, biophysical analysis, and anti-infective microbiology, benefiting from the expertise of my co-mentors, Drs. Richard Lee and Jason Rosch, and the state-of-the-art resources at St. Jude.