Browsing by Subject "Inhibitors"
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Item Influence of autoinducer 2 (ai-2) and ai-2-like inhibitors generated from ground beef on escherichia coli o157:h7 protein expression(2009-05-15) Soni, Kamleshkumar A.Autoinducer 2 (AI-2) molecules produced by bacterial cells are thought to be involved in controlling a variety of bacterial cellular processes by coordinated gene and protein expression. Previous work in our laboratory has shown that ground beef contains compounds that can interfere with AI-2-mediated bioluminescence expression in Vibrio. harveyi. The underlying hypothesis of this work was that AI-2 molecules affect the protein expression in Escherichia coli O157:H7 and AI-2 inhibitory molecules negate the influence of AI-2 molecules. The main objectives of this study were to identify, characterize, and isolate the factors responsible for inhibition of AI-2 molecules from ground beef extracts, elucidate the role of LuxS/AI-2 cell signaling system in E. coli O157:H7 protein expression, and determine if inhibitory factors present in ground beef extract can negate the influence of AI-2 molecules on the protein expression. Using a solvent extraction procedure and gas chromatography analysis, AI-2 inhibitory factors present in ground beef extracts were identified as both medium and long chain fatty acids. When identified fatty acids were tested at different concentrations for AI-2 inhibition, AI-2 inhibition ranging from 25% to 90% was observed. Both ground beef extracts and mixture of selected fatty acids also resulted in 2- to 4-fold reduced AI-2 influenced biofilm formation by E. coli K12 cells. Identification of LuxS/AI-2-mediated protein expression in E. coli O157:H7 was conducted using two dimensional gel electrophoresis. Protein expression analysis showed that the LuxS/AI-2 system modulates the expression of proteins involved in different cellular processes such as carbohydrate and amino acid metabolism, stress response, and formation of flagella and motility. When AI-2 inhibitory factors were added along with AI-2 molecules, the expression patterns of three AI-2-influenced proteins (GlmS, SpeE, and NikA) were changed suggesting that AI-2 inhibitors can negate the influence of AI-2 molecules on protein expression of selected proteins. Collectively, these results highlight that proteins associated with different cellular processes in E. coli O157:H7 can be modulated depending on whether cells are in contact with AI-2 molecules in the presence or absence of AI-2 inhibitory factors.Item Progress in the search for ricin A chain and shiga toxin inhibitors(2009-12) Bai, Yan, 1977-; Robertus, Jon D.Ricin and Shiga toxin type 1 are potent cytotoxins known as ribosome inhibition proteins, abbreviated RIPs. Proteins of this family shut down protein synthesis by removing a critical adenine in the conserved stem-loop structure of 28S rRNA. Due to its exquisite cytotoxicity, the plant toxin ricin has been used as a biological warfare agent. Although great achievement has been made on ricin research, including catalytic mechanism and structure analysis, there is still no specific treatment available for ricin exposure. In addition, ricin A chain inhibitors may also be useful against the homologous bacterial proteins shiga toxins, which are responsible for dysentery, and diseases related to food poisoning, including hemolytic uremic syndrome. Previous study on RTA inhibitor search has provided a number of substrate analog inhibitors, all of which, however, are weaker inhibitors. Therefore, the goal of this work is to improve the binding affinity of known inhibitors and to discovery new scaffolds for inhibitor discovery and development. In this work, multiple approaches were employed for this purpose, including optimizing known inhibitors and searching new inhibitors by Virtual Drug Screening (VDS) and High Throughput Screening (HTS). A number of new RTA inhibitors were discovered by these strategies, which provide a variety of pharmacophores for RTA inhibitor design, and also added a new line of evidence for VDS as an advanced technology for drug discovery and development.Item Rational design and syntheis of substrate-based inhibitors of steroidogenesis(Texas Tech University, 2008-12) Liu, Jialin; Nes, William David; Shaw, Robert W.; Weber, JoachimSterol biosynthesis is crucial to all groups of life forms. Targeted inhibition of this pathway by substrate-based analogs is currently used to probe sterol function and may have therapeutic importance. In this dissertation, a series of new steroidal triperpenes with a modified lanosterol or cycloartenol frame have been designed to inhibit steroidogenesis. These compounds, along with a number of known sterol biosynthesis inhibitors with the cholestane skeleton, have been prepared and characterized in detail using GC, MS, HPLC and NMR. Notably, a series of substrate analogs constructed with a methyl, nitrogen, sulfur, bromine or fluorine atom or altered to possess a methylene cyclopropane, or elongated to contain a terminal double or triple bonds were prepared to act as mechanism-based inactivators of the sterol 24-methyl transferase enzyme. In addition, compounds with the cholestane and lanostane structures were prepared with modifications at C-7 and C-32 to be reversible and irreversible inhibitors of the ¦¤8- ¦¤7-isomerase and 14¦Á-demethylase enzymes, respectively.Item Structural Studies and Evaluation of Inhibitors of Mycobacterium tuberculosis H37Rv Shikimate Dehydrogenase (MtSDH)(2014-03-21) Lalgondar, MallikarjunShikimate dehydrogenase (SDH) is a reversible enzyme catalyzing the reduction of 3-dehydroshikimate (3DHS) to shikimate (SKM) utilizing NADPH cofactor in the shikimate pathway, a central route for biosynthesis of aromatic amino acids, folates and ubiquinones in microogransims, plants and parasites, which renders the enzymes of this essential pathway as attractive targets for developing antimicrobials, herbicides and antiparasitic agents. In this study, the crystal structure of Mycobacterium tuberculosis SDH (MtSDH) was determined in the apo-form and in complex with a ligand, SKM. The overall structure of MtSDH contains two structural domains with ?/? architecture. The N-terminal substrate binding domain and C-terminal cofactor binding domain are interconnected by two helices forming an active site groove where catalysis occurs. In MtSDH, a series of helices connecting ?10 and ?11 strands replace a long loop found in other known SDH structures and this region may undergo structural changes upon cofactor binding. NADP^(+) was modeled reliably in the cofactor binding site to gain insight into specific interactions. The analysis reveals that NADP(H) binds in anti conformation and in addition to residues in ?basic patch?, Ser125 within the glycine rich loop may interact with the 2'-phosphate of adenine ribose and form a novel cofactor binding microenvironment in SDH family of enzymes. Biochemically, five inhibitors identified previously from a high-throughput enzyme assay screen were evaluated. The IC_(50) values of these compounds range from 2.8-4.6 ?M. Further investigation indicates that these compounds display non-competitive or mixed inhibition mode with both substrate and cofactor. This study is expected to provide better understanding of MtSDH structural features and a framework for rational design of inhibitors based on initially characterized compounds.Item Synthesis and optimization of a library of small molecule inhibitors of ricin toxin A(2012-05) Pruet, Jeffrey Michael; Anslyn, Eric V., 1960-Ricin is a potent cyctotoxin with no known antidote. Chapter 1 provides background and context for this thesis, which is primarily focused on probing the active site of Ricin toxin A (RTA). Relevant information about Ricin, its use, method of action, and noteworthy contributions towards the discovery of Ricin A chain inhibitors are provided. Furthermore, a brief description of the assays used by our collaborators to monitor RTA inhibition is provided. Additionally, a great deal of this thesis pertains to a particular heterocycle, pterin, and thus the remainder of Chapter 1 is dedicated to pterins, their physical properties, biological relevance, and selected reports of pterin chemistry. Chapter 2 details preliminary research focused on the use of nucleic acid-based platforms as RTA inhibitors. Two specific nucleic acids were chosen, adenine and guanine, and the chapter is split to address them individually. Rational for their use is provided, as well as the synthetic strategies investigated. Both platforms showed significant interference with the analysis assay, most pronounced for the adenine series. A primary goal throughout this thesis is the identification of a simple, rapid method to provide a library of new compounds. To this end, discussion of improved synthetic routes are provided within the section dedicated to guanines. Initial investigation into pterins as a platform for RTA inhibitors is provided in Chapter 3. Much of this chapter is concerned with hurtles encountered while dealing with the poor solubility of pterins, purification, and limits in reaction scope. Finally this chapter details a significant discovery in pterin's utility, both in terms of synthetic ease and preference towards one regioisomer over another. A variety of amides are initially used to probe the active site for significant interactions to the pterin pendents. Chapter 4 builds off the discoveries detailed within the previous chapter. Efforts to optimize the preliminary amide series from Chapter 3 are described, leading to a significant enhancement in activity. Additionally, Chapter 4 describes a synthetic breakthrough which greatly enhanced the speed of synthesis and complexity of the designed pterin inhibitors. Building upon the goal to map the RTA active site, a description of various peptide conjugated pterins is provided, as well as efforts to arrive at optimized isosteres of the most promising peptide derivatives.