Use of a novel tricyclic Stk1 inhibitor to uncover molecular mechanism in clinically relevant strains of S. aureus

项目摘要/摘要-希瑟·米勒博士 蛋白激酶Stk1控制着与抗生素耐药性、生物膜形成和 毒素在金黄色葡萄球菌强毒株中的表达。迫切需要确定 它的作用机制。如果没有这些信息，就会缺少关于一个重要的 将抗生素耐药性降至最低的工具。体外抑制STK1增强甲氧西林耐药 金黄色葡萄球菌(MRSA)对β-内酰胺类抗生素的耐药性，使其成为具有吸引力的开发目标 新型抗生素佐剂。然而，对stk1缺失突变的研究产生了相互矛盾的结果。 关于这种蛋白质在毒力中的作用的结果。被认为是主要监管者，很难预测 STK1抑制的全部后果。此外，下游菌株的特定差异 受Stk1磷酸化影响的基因表达阻碍了对 在医学上相关的背景下。目前还不清楚STK1抑制剂是如何诱导这些抗生素佐剂的 效果。这项研究的长期目标是开发新的抗毒力疗法来对抗 持续性和抗药性细菌感染。我们的总体目标是确认 金黄色葡萄球菌先导化合物的分子靶点及其对Stk1抑制作用的临床研究 相关的MRSA菌株，并开发基因工具来解码STK-1的机制细节 介导性β-内酰胺类耐药。我们的中心假设是我们的先导化合物代表一种 有希望的抗生素佐剂支架，目标是主调节剂Stk1。为了测试这一点 假设，佐剂将被用作询问Stk1结构域的化学探针(S) 对捆绑是必要的。比较转录组学将被用来研究Stk1介导的 几种MRSA菌株的基因表达以阐明可能影响的差异 开发具有广泛活性的抑制剂。最后，基因工具将被开发出来，将 系统突变磷酸化底物追踪Stk1介导的β-内酰胺耐药 这些临床相关耐甲氧西林金黄色葡萄球菌的通路。在完成建议的工作后，我们 期望对现有的毒力途径及其调控知识做出贡献 金黄色葡萄球菌的机制。这项工作是创新的，因为实验设计没有 坚持现状不变。我们不仅要研究整个转录组的临床变化 相关菌株，但也与佐剂分子单独和与抗生素联合作用 提供一个更具医学相关性的背景。这些结果将产生重大影响，因为它们将 为评估Stk1作为治疗靶点提供必要的信息，从而提供 为开发新的抗菌疗法提供关键的新信息。

Project Summary/Abstract – Dr. Heather Miller, PI The kinase Stk1 controls genes involved in antibiotic resistance, biofilm formation, and toxin expression in virulent strains of Staphylococcus aureus. There is a critical need to determine its mechanism of action. Without this information, there are key details missing about an important tool in minimizing antibiotic resistance. Inhibition of Stk1 in vitro potentiates methicillin-resistant S. aureus (MRSA) to b-lactam antibiotics, making it an attractive target for the development of novel antibiotic adjuvants. However, studies of stk1 deletion mutants have produced contradictory results about this protein’s role in virulence. Considered a master regulator, it is difficult to predict the full consequences of Stk1 inhibition. Additionally, the strain-specific differences in downstream gene expression affected by Stk1 phosphorylation have prevented comprehensive understanding in a medically relevant context. It is not clear yet how Stk1 inhibitors elicit these antibiotic adjuvant effects. The long-term goal of this research is to develop novel anti-virulence treatments to combat persistent and antibiotic resistant bacterial infections. Our overall objectives are to confirm the molecular target of the lead compound in S. aureus, investigate this Stk1 inhibition in clinically relevant strains of MRSA, and develop genetic tools to decode mechanistic details of Stk-1 mediated b-lactam resistance. Our central hypothesis is that our lead compound represents a promising antibiotic adjuvant scaffold that targets the master regulator Stk1. To test this hypothesis, adjuvants will be used as chemical probes to interrogate the Stk1 domain(s) necessary for binding. Comparative transcriptomics will be used to investigate Stk1-mediated gene expression across several strains of MRSA to elucidate differences that could affect development of broadly active inhibitors. Finally, genetic tools will be developed that will systematically mutate phosphorylated substrates to trace Stk1-mediated b-lactam resistance pathways in these clinically relevant MRSA strains. Upon completion of the proposed work, we expect to contribute to the existing knowledge of virulence pathways and their regulatory mechanisms in S. aureus. This work is innovative because the experimental design does not adhere to the status quo. We will not only investigate transcriptome-wide changes across clinically relevant strains, but also with adjuvant molecules alone and in combination with antibiotics to provide a more medically relevant context. These results will have a significant impact as they will afford necessary information for evaluation of Stk1 as a therapeutic target, thereby providing crucial new information for the development of novel antibacterial therapies.