R15 AREA: Optimizing allosteric modulation of noncoding regulatory RNA function

点击翻译按钮获取中文摘要

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.