Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item Intercepting Cyclic Dinucleotide Signaling with Small Molecules(2016) Zheng, Yue; Sintim, Herman O; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial infections, especially the ones that are caused by multidrug-resistant strains, are becoming increasingly difficult to treat and put enormous stress on healthcare systems. Recently President Obama announced a new initiative to combat the growing problem of antibiotic resistance. New types of antibiotic drugs are always in need to catch up with the rapid speed of bacterial drug-resistance acquisition. Bacterial second messengers, cyclic dinucleotides, play important roles in signal transduction and therefore are currently generating great buzz in the microbiology community because it is believed that small molecules that inhibit cyclic dinucleotide signaling could become next-generation antibacterial agents. The first identified cyclic dinucleotide, c-di-GMP, has now been shown to regulate a large number of processes, such as virulence, biofilm formation, cell cycle, quorum sensing, etc. Recently, another cyclic dinucleotide, c-di-AMP, has emerged as a regulator of key processes in Gram-positive and mycobacteria. C-di-AMP is now known to regulate DNA damage sensing, fatty acid synthesis, potassium ion transport, cell wall homeostasis and host type I interferon response induction. Due to the central roles that cyclic dinucleotides play in bacteria, we are interested in small molecules that intercept cyclic dinucleotide signaling with the hope that these molecules would help us learn more details about cyclic dinucleotide signaling or could be used to inhibit bacterial viability or virulence. This dissertation documents the development of several small molecule inhibitors of a cyclic dinucleotide synthase (DisA from B. subtilis) and phosphodiesterases (RocR from P. aeruginosa and CdnP from M. tuberculosis). We also demonstrate that an inhibitor of RocR PDE can inhibit bacterial swarming motility, which is a virulence factor.Item NEW CHEMICAL TOOLS TO INVESTIGATE RNA FUNCTIONS(2013) Luo, Yiling; Dayie, Theodore K; Sintim, Herman O; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribonucleic acid (RNA), as one of the essential macromolecules of life, plays an active role in gene regulation, catalysis, and signaling because of its ability to adopt complex 3D structures that can exist in multiple conformations. Until now, RNA preparation methods devised by most investigators utilized partially denatured the RNA. The mis-folding caused by denaturing - renaturing can seriously affect RNA structure and functional activity. To test this hypothesis, in PART I of this dissertation, we presented a simple strategy using `click' chemistry to couple biotin to a `caged' photocleavable guanosine monophosphate to synthesize native RNAs that are properly folded. We demonstrated that RNA ribozymes, ranging in size from 27 to 527 nt, prepared by our non-denaturing method form a homogenous population with superior catalytic activity than those prepared by traditional refolding methods. Having developed a method for in vitro RNA synthesis, we studied the riboswitch family of RNAs that require remolding their structure for function in PART II. C-di-GMP riboswitch, as the only currently known secondary messenger riboswitch, senses c-di-GMP using its aptamer domain to modulate the expression of genes which affects biofilm formation and virulence factors production in bacteria. To specifically target pathogenic bacteria in polymicrobial systems that control the RNA-mediated c-di-GMP signaling pathway through riboswitch regulation, different c-di-GMP analogs have been used as chemical tools to investigate the structure-activity relationship (SAR) of c-di-GMP binding to different c-di-GMP riboswitches. We demonstrated that different 2'-position modified c-di-GMP analogs could differentiate between two different classes of c-di-GMP riboswitches and even bind to one particular riboswitch in class I with different affinities. Specifically, Clostridium tetani (Ct-E88) RNA in class I c-di-GMP riboswitch was used for (structural dynamic) study to understand how ligand binding drives the conformational change to regulate the downstream genes within the expression platform. Our preliminary data obtained via different biochemical and biophysical tools - such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy - demonstrated that Mg2+ ions accelerate ligand recognition by pre-organizing the RNA, and then rapid ligand binding folds the RNA into a compact structure for likely downstream gene regulation.Item New molecules to combat the bacterial antibiotic resistance problem(2011) WANG, JINGXIN; Sintim, Herman O; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Platensimycin/platencin are recently discovered natural products that inhibit membrane formation in bacteria and cyclic diguanylic acid (c-di-GMP) is a master regulator of bacterial biofilm formation. The rise of bacterial antibiotic resistance and the dwindling pipeline of new antibiotics make these molecules of interest to the scientific community. This dissertation reports the design, synthesis and biological evaluation of analogs of platensimycin/platencin and c-di-GMP. Platensimycin and platencin have garnered interest from synthetic chemists due to the complexity of their molecular architecture, coupled with their exciting biological profile (inhibition of bacterial fatty acid synthases). We have developed a concise synthetic approach towards the platensimycin/platencin class of antibiotics. The highlight of our synthesis is the use of dynamic ring-closing metathesis to prepare a bicyclo intermediate and a tandem nucleophilic addition of organolithium to a ketone moiety, followed by a subsequent ring opening of a nearby epoxide to generate complex tricyclic framework. The synthesis of platensimycin or closely related analogs requires multi-steps (average of 17 overall steps). Using a function-oriented synthetic approach, we developed short syntheses of N,N-dialkyl benzoic acid derivatives of platensimycin, and we demonstrate that these readily prepared molecules have comparable antibiotic properties to the difficult-to-synthesize platensimycin/platencin. C-di-GMP has been dubbed the master regulator of bacterial "lifestyle" due to the key role that this molecule plays in bacterial biofilm formation and virulence formation. In order to study c-di-GMP signaling in bacteria, with the ultimate goal of using key insights gained from such studies to develop anti-biofilm or anti-virulence agents, we prepared analogs of c-di-GMP and studied their biophysical and biological profiles. Interestingly, we reveal that conservative modifications to c-di-GMP affect both the biophysical and biochemical properties of this molecule. We also demonstrate a concept called "conformational steering" as a powerful principle to selectively target different classes of receptor proteins that bind to c-di-GMP.