R15 AREA: Optimizing allosteric modulation of noncoding regulatory RNA function

项目摘要 细菌耐药性是一个重大的全球性健康威胁， 细菌感染（包括革兰氏阳性菌感染）转化为致命疾病。为了克服这个 严重威胁人类健康，需要全新的抗生素。T盒核糖开关是 一种非编码RNA，调节许多革兰氏阳性细胞中必需基因的表达， 细菌（包括致病性实例），从而使其成为新型抗菌药物的独特靶标。 剂. T-box核糖开关调控机制的一个关键组成部分涉及tRNA结合 到高度保守的抗终止子RNA元件--一个很好的药物发现靶点。这 项目将探索T-box抗终止子RNA变构调节剂的合理设计 功能并将其功效与竞争性抑制剂的功效进行比较。没有治疗剂是 目前已知特异性靶向T盒核糖开关。 我们先前的研究表明，核糖开关抑制可以通过负变构发生， 抗终止子的调节而不是与核糖开关的同源物的直接竞争 配体（tRNA）。因此，该提议寻求鉴定具有改善的活性的化合物 基于以下假设：利用变构调节是有效的药物 针对非编码RNA功能的发现策略。具体来说，我们计划使用 设计下一代抑制T-box核糖开关化合物的计算方法 功能将开发优化的高通量筛选方案，包括 诱导拟合对接和精细评分方法。我们将合成三组不同的 在初步RNA研究中显示出活性的化合物或已经设计的化合物， 通过最初的计算研究。将对选定的化合物进行表征， 核糖开关抑制浓度（IC 50），抗终止剂结合（选择性，Kd），RNA结合 模式和最小抑制浓度。 实现拟议的具体目标将通过发展 设计策略以鉴定RNA功能的抑制剂（包括变构调节剂）， 探索这些小分子最有效的化学空间特征。这些信息 将对靶向T-box核糖开关的抗菌药物发现以及 靶向非编码调节RNA的更广泛领域仍在出现， 抗病毒、抗癌和其他药物发现的努力。在这个过程中，一个主要由 本科生将获得第一手的经验，对创新药物的发现工作 该项目将激励他们考虑从事生物医学研究。

Project Summary Bacterial drug resistance is a significant global health threat that has turned once treatable bacterial infections (including Gram-positive infections) into deadly illnesses. To overcome this serious threat to human health, completely novel antibiotics are needed. The T-box riboswitch is a noncoding RNA that regulates the expression of essential genes in many Gram-positive bacteria (including pathogenic examples), thus making it a unique target for novel antibacterial agents. A key component of the T-box riboswitch regulatory mechanism involves tRNA binding to the highly conserved antiterminator RNA element--an excellent target for drug discovery. This project will explore the rational design of allosteric modulators of T-box antiterminator RNA function and compare their efficacy to that of competitive inhibitors. No therapeutic agents are currently known to specifically target the T-box riboswitch. Our prior studies indicate that riboswitch inhibition can occur through negative allosteric modulation of the antiterminator rather than direct competition with the riboswitch’s cognate ligand (tRNA). Consequently, this proposal seeks to identify compounds with improved activity based on the following hypothesis: Leveraging allosteric modulation is an effective drug discovery strategy for targeting noncoding RNA function. Specifically, we plan to use computational methods to design the next generation of compounds that inhibit T-box riboswitch function. An optimized high throughput screening protocol will be developed that includes induced-fit docking and refined scoring methods. We will synthesize sets of 3 different compounds that have either shown activity in preliminary RNA studies or have been designed through initial computational studies. Select compounds will be characterized to quantify riboswitch inhibitory concentration (IC50), antiterminator binding (selectivity, Kd), RNA binding mode, and minimum inhibitory concentrations. Achievement of the proposed specific aims will improve scientific knowledge by developing design strategies to identify inhibitors (including allosteric modulators) of RNA function and by exploring the most effective chemical space features of these small molecules. This information will be significant for antibacterial drug discovery targeting the T-box riboswitch and also for the still emerging broader field of targeting noncoding regulatory RNA in general for antibacterial, antiviral, anticancer and other drug discovery efforts. In the process, a team of primarily undergraduate students will gain first-hand experience working on an innovative drug discovery project that will inspire them to consider careers in biomedical research.