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

项目总结/摘要-石楠米勒博士，PI 激酶Stk 1控制涉及抗生素耐药性、生物膜形成和 金黄色葡萄球菌强毒株的毒素表达。现在迫切需要确定 其作用机制。如果没有这些信息， 减少抗生素耐药性的工具。体外抑制Stk 1增强甲氧西林耐药性 S.金黄色葡萄球菌（MRSA）对b-内酰胺抗生素的敏感性，使其成为开发 新的抗生素佐剂。然而，对stk 1缺失突变体的研究产生了矛盾的结果。 关于这种蛋白质在毒力中的作用的结果。被认为是一个主要的监管机构，很难预测 Stk 1抑制的全部后果。此外，下游的菌株特异性差异 受Stk 1磷酸化影响的基因表达阻碍了对 在医学相关的背景下。目前尚不清楚Stk 1抑制剂如何引起这些抗生素佐剂 方面的影响.这项研究的长期目标是开发新的抗病毒治疗方法， 持续性和抗生素耐药性细菌感染。我们的总体目标是确认 S.金黄色葡萄球菌，在临床上研究Stk 1抑制作用 MRSA相关菌株，并开发遗传工具来解码Stk-1的机制细节 介导的β-内酰胺抗性。我们的中心假设是，我们的先导化合物代表了一种 靶向主调节因子Stk 1的有前途的抗生素佐剂支架。为了验证这一 假设，佐剂将用作化学探针来询问Stk 1结构域 需要绑定。比较转录组学将用于研究Stk 1介导的 几种MRSA菌株的基因表达，以阐明可能影响 开发具有广泛活性的抑制剂。最后，将开发遗传工具， 系统性突变磷酸化底物以追踪Stk 1介导的b-内酰胺抗性 在这些临床相关的MRSA菌株中的途径。在完成拟议工作后，我们 希望有助于现有的知识毒力途径及其调控 S.金黄色。这项工作是创新的，因为实验设计不 坚持现状。我们不仅将研究临床上的转录组变化， 相关菌株，但也可以单独使用佐剂分子和与抗生素组合使用佐剂分子， 提供了一个更医学相关的背景。这些结果将产生重大影响，因为它们将 为评估Stk 1作为治疗靶点提供必要的信息，从而提供 为新型抗菌疗法的发展提供了重要的新信息。

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.