Browsing by Author "Cohen, Robert, committee member"
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Item Open Access Adenosine triphosphate is an allosteric inhibitor of coxsackievirus B3 3Dpol(Colorado State University. Libraries, 2016) Karr, Jonathan Paul, author; Peersen, Olve, advisor; Cohen, Robert, committee member; Perera, Rushika, committee memberPicornaviruses pose a significant threat to human and animal health, but at present there are no drugs to prevent or treat picornaviral infections. However, intensive study of the picornaviral lifecycle has revealed several promising pharmacological targets, including the RNA-dependent RNA polymerase, 3Dpol, that is responsible for replicating the viral genome. 3Dpol is central in the virus lifecycle, determines the distribution of mutants in viral progeny, and is a very highly conserved protein across picornavirus species. As such, it is an attractive target for antiviral research. The only 3Dpol inhibitors that have been found to date are nucleoside analogues that act directly on the active site, even though global dynamics of the protein that are sensitive to allosteric effects of various mutations have been shown to be important determinants of fidelity. The research presented in this thesis provides the first direct evidence of allosteric regulation of 3Dpol by a small molecule. Inhibition assays investigating the relative affinities of a stalled coxsackievirus elongation complex for non-cognate nucleotides uncovered a mixed inhibition profile of ATP. Among the six picornavirus species tested, this mode of inhibition seems specific to coxsackievirus B3 (CVB3). Engineered mutations in CVB3 3Dpol, including two that were previously found to lower polymerase fidelity, diminish the uncompetitive component of ATP inhibition. ATP inhibition was found to be dependent on the β- and γ-phosphates. The potential role of ATP’s allosteric effect in the virus lifecycle as well as the importance of a biochemically confirmed allosteric site on the polymerase are discussed.Item Embargo Advancing the utility of organic superbases in synthetic methodology(Colorado State University. Libraries, 2023) Sujansky, Stephen J., author; Bandar, Jeffrey, advisor; Miyake, Garret, committee member; Sambur, Justin, committee member; Cohen, Robert, committee memberDeprotonation is one of the most fundamental and important modes of molecular activation, making Brønsted bases a critical part of a synthetic chemist's toolbox. An exceptional class of Brønsted bases are organic superbases, which are finding increased use in modern synthetic methods due to their unique properties. This thesis describes the use of these unique properties to advance the synthetic utility of superbases in two ways; 1) improving superbase- catalyzed alkene hydrofunctionalization reactions; and 2) developing air-stable and convenient organic superbase prereagents. Chapter One describes organic superbases in detail to provide background and context for Chapters Two and Three. Within Chapter One, various classes of superbases are presented, as well as their unique properties, syntheses, and example applications. Finally, the limitations and challenges associated with the use of superbases are discussed. Chapter Two describes the Bandar Group's superbase-catalyzed alkene hydrofunctionalization methodology. Within this chapter mechanistic studies as well as computational modeling, done as part of a collaboration with the Paton Group, are presented. These mechanistic studies provided insight into the factors controlling the reaction equilibrium. This insight was then used to logically address the limitations associated with the original conditions reported by the Bandar Group in 2018. The results of this work help to improve reaction efficiency and to expanded substrate scope. This understanding also led to the development of a catalytic anti-Markovnikov aryl alkene hydration method that allows convenient access to β-aryl alcohols. Chapter Three describes the development of air-stable organic superbase precatalysts and prereagents. Superbase salts that decarboxylate were developed as a first strategy method to generate the neutral superbase in solution. This initial salt system then led to the discovery of stable superbase carboxylate salts that react with and open epoxide additives in situ to neutralize the superbase conjugate acid. This ring strain release strategy is shown to be effective at promoting a range of reactions including Michael-type addition, ester amidation, deoxyfluorination, SNAr and Pd-catalyzed cross coupling reactions. These superbase precatalysts and prereagents provide a means to access the unique properties of organic superbases from air-stable and easy-to-handle salts. Overall, Chapters Two and Three represent significant progress in advancing the utility of organic superbases in synthetic methodology. My work in Chapter Two, along with the Bandar's and Paton Group's efforts, meaningfully expanded the scope and usefulness of superbase- catalyzed alcohol addition reactions. Our new mechanistic understanding proved to be fundamental to a range of addition reactions and pushed the boundary of possible nucleophilic addition reactions. My efforts in Chapter Three, along with Garrett's significant contributions, have made organic superbase much more convenient to use, synthesize and store. This greater convenience and potentially lower cost can be expected to improve access to superbase chemistry and serve as the foundation for future discoveries. Additionally, the ability to control the concentration of superbase in solution will have many benefits in expanding substrate scopes and modulating reaction profiles where a strong base is required but is also detrimental to the overall process.Item Open Access Characterization and quantification of urinary metabolic biomarkers for early response to anti-tuberculosis treatment(Colorado State University. Libraries, 2016) Fitzgerald, Bryna, author; Belisle, John, advisor; Crick, Dean, committee member; Dobos, Karen, committee member; Cohen, Robert, committee memberDevelopment of new anti-tuberculosis (TB) therapies remains a major priority to combat this infectious disease and to prevent continued transmission of the causative agent Mycobacterium tuberculosis (Mtb). However, newly developed therapies require large, lengthy clinical trials to determine the number of treatment failures and relapses for evaluation of treatment efficacy. Biomarkers for the prediction of treatment outcome in TB patients at early time points would facilitate movement of new therapies through clinical trials. Previously, liquid chromatography-mass spectrometry (LC-MS) based metabolomics experiments identified potential biomarkers for early response to anti-TB treatment. The research presented in this dissertation involves experiments needed for the progression of these compounds towards a clinically useful biosignature. A major impediment to metabolomics-based biomarker discovery is metabolite identification, as approximately 50% of detectable products do not match structures in existing databases. In concordance with this, several of the potential small molecule biomarkers of anti-TB treatment response lacked structural identification. This research resulted in the structural characterization of three of these compounds as a core 1 O-glycosylated SerLeu peptide, N-acetylisoputreanine, and N1, N12-diacetylspermine. Both the core 1 O-glycosylated SerLeu peptide and N-acetylisoputreanine are novel compounds that had not been previously detected in human urine. Characterization of these compounds indicated a potential alteration of polyamine catabolism and the complement and coagulation pathways during anti-TB treatment. Another key aspect in biomarker discovery is defining the processes involved in formation of potential biomarkers. In order to determine whether these compounds were formed by processes upregulated during active disease, the abundances of these compounds were assessed in active TB patients and household contacts as well as in Mtb infected and uninfected Balb/c mice. The core 1 O-glycosylated SerLeu peptide and N1, N12-diacetylspermine were increased in the urine of index patients demonstrating a potential link between Mtb infection, associated disease pathology, and the formation of these compounds. N-acetylisoputreanine, however, was not increased in TB patient urine or infected mouse tissue indicating that this compound may be formed due to off target drug interactions. These experiments not only provided insights into the mechanisms behind alteration of these compounds during anti-TB treatment, but also highlighted those compounds that may be better biomarkers for anti-TB treatment response. Assessment of these compounds using an independent set of patient samples is needed to validate them as biomarkers for early anti-TB treatment response. Unlike the untargeted experiments used for discovery of potential biomarkers, validation typically employs targeted assays. This research describes the development of a targeted multiple reaction monitoring (MRM) assay which enabled accurate and precise quantitation of compounds previously detected in an untargeted metabolomics experiment. This targeted assay will be used for validation of these compounds in a larger set of patient samples representing a variety of different treatment outcomes. Overall these experiments confirmed the identity of three metabolites that decrease with anti-TB treatment response. Two of these metabolites are novel compounds and their characterization adds to metabolite databases expanding the number of metabolites available to other metabolomics researchers. Assessment of these compounds in samples representative of active TB disease confirmed two of them as promising biomarkers for anti-TB treatment response and highlighted another as a potential result of unintended drug effects. The development of a MRM assay for the quantification of these compounds enables their validation and confirmation as biomarkers of anti-TB treatment response. The work presented in this dissertation describes the advancement of metabolites identified during biomarker discovery towards application in clinical trials.Item Open Access Characterization of the selective hydrolysis of branched ubiquitin chains by Uch37 and its activator Rpn13(Colorado State University. Libraries, 2020) Hazlett, Zachary S., author; Yao, Tingting, advisor; Cohen, Robert, committee member; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Kennan, Alan, committee memberThe ubiquitin (Ub) C-terminal hydrolase, Uch37, can be found associated with the 26S proteasome as well as the INO80 chromatin remodeling complex. Bound to the 26S proteasome, it assists in regulating the degradation of Ub modified proteins. The proteasomal subunit Rpn13 binds Uch37, anchors it to the proteasome 19S regulatory particle and enhances the deubiquitinating enzyme's (DUB's) catalytic activity. While the structure of the Uch37/Rpn13 complex bound to a single Ub molecule has been characterized, much still remains unknown regarding the enzyme's substrate specificity, the molecular basis for its substrate specificity, and its function in the regulation of proteasomal degradation. In this thesis we characterize the substrate specificity of Uch37 with and without its proteasomal binding partner Rpn13. By synthesizing poly-Ub chains of various linkage types and topologies and using these Ub chains in in vitro deubiquitination assays, we were able to determine that Uch37/Rpn13 selectively cleaves branched Ub chains. This provides evidence to suggest that Uch37 is the first enzyme with activity specific for branched Ub chains. Branched Ub chains have been identified endogenously and have roles connected to the regulation of nascent misfolded polypeptides, cell cycle control, and the enhancement of proteasomal degradation. The work presented here sets out to characterize the molecular mechanism of branched chain hydrolysis by Uch37 and its binding partner Rpn13, determine the kinetics of this enzymatic reaction, and establish a system for probing the function of "debranching" by Uch37 in proteasomal degradation. The conclusion of our work builds our understanding of the complex system of intracellular signaling by Ub and unveils key elements to the primary system responsible for regulating cellular protein homeostasis.Item Open Access Development of computational tools to model molecular interactions for medicinal chemistry(Colorado State University. Libraries, 2017) Ford, Melissa Coates, author; Ho, P. Shing, advisor; Cohen, Robert, committee member; Snow, Christopher, committee member; McCullagh, Martin, committee memberMedicinal chemistry has evolved over the past 40 years to rely heavily on the computationally aided design of new drugs. The work in this dissertation focuses on developing computational tools for the application of medicinal chemistry. For computational techniques to be dependable, important interactions must be properly modeled and the techniques must be rigorously tested. In this work, I first introduce an important interaction for drug design, the halogen bond (X-bond). We consider how decades of work has come closer to properly modeling the X-bond, yet there remain many unexplored areas. Two areas are addressed in this dissertation: the structure-energy relationship of 1) a Br…S- X-bond in a DNA junction and 2) Br…O and I…O X-bonds in T4 Lysozyme (T4-L). Using these systems, we can better understand the X-bond and further test computational tools. One such tool, a molecular mechanics/dynamics package, TINKER, does not model X-bonds. Thus, I then incorporate a force field for a broad range of X-bonding molecules into TINKER, creating X-TINKER. X-TINKER reproduces the energies and geometries of the X-bond in the DNA and T4-L systems. Last, I will discuss testing a different software developed by Schrödinger, FEP+. We find FEP+ can effectively predict protein stability; however, it still has areas that need improvement. Together, the findings of this dissertation emphasize the importance of understanding molecular interactions, improving algorithms, and testing current programs to find remaining failures. By continuing to use this cycle, we hope to see the impact of computational tools in medicinal chemistry.Item Open Access Long range interaction networks within 3Dpol and the roles they play in picornavirus genome replication and recombination(Colorado State University. Libraries, 2020) Watkins, Colleen L., author; Peersen, Olve B., advisor; Cohen, Robert, committee member; Ho, P. Shing, committee member; Wilusz, Jeffrey, committee memberPicornaviruses contain a single-stranded positive sense RNA genome approximately 7.5kb in length. The genome encodes for a single polyprotein that can future be divided into three functional regions; the P1 region containing the viral capsid proteins, the P2 region whose proteins function primarily in membrane rearrangement during viral replication, and the P3 region which contains four protein responsible for RNA replication. The final protein in the P3 region is 3Dpol, an RNA-dependent RNA polymerase (RdRP) whose structure is analogous to a "right hand" with fingers, palm and thumb domains, and around which this dissertation will be centered. Section one of this work investigates the roles three regions within the fingers domain play in the catalytic cycle of 3Dpol: "The kink" located within the index finger, "the gateway" found on the pinky, and "the sensor", which bridges the two beta-strands of the middle finger. This study demonstrates that the kink residues are involved in RNA binding as mutations to these residues result in decreased initiation time and elongation complex lifetime. The gateway residues are found to act as a molecular stop against which the template-RNA strand positions itself post-translocation, eventually resetting the active site for the next round of nucleotide incorporation. Lastly the sensor residues serve two key functions: 1) A final checkpoint to determine the correct nucleotide has entered the active site, and 2) As a possible source for proton donation to the pyrophosphate leaving group formed during catalysis. The inter-connected nature of the residues investigated in this section give rise to the idea that it is not individual residues alone that control major steps during the catalytic cycle, but instead that long ranging interaction networks within the different polymerase domains are ultimately responsible for controlling different actions carried out by the polymerase. Section two of this work looks at the long-range interaction networks within 3Dpol by dissecting the roles each polymerase domain plays in catalytic cycle. Through generation of chimeric polymerases it was determined that the pinky finger, with some influence by the fingers domain, controls RNA binding, the palm domain dictates nucleotide discrimination, and nucleotide capture and active site closure rates. It was also established that the thumb domain controls translocation, and an interaction between the palm and thumb domains was needed to generate a viable virus, supporting the idea of interface I, a protein-protein interface that was discovered in the first 3Dpol crystal structure. What is most striking about these findings is that unlike other single subunit polymerases that perform translocation by using a large swinging motion within the fingers domain, viral RdRPs use an entirely different domain altogether. The last section of this work deals with viral recombination, an event that is carried out at a low frequency during virus replication. Recombination is proposed to be a mechanism by which mutations can be purged from the genome independent of polymerase fidelity. This study carries out a mechanistic investigation into how mutation of residue 420 from a leucine to an alanine affects polymerase replication kinetics. It also takes a look at the mutation of residue 64 from a glycine to a serine, a previously identified mutation that results in a high-fidelity polymerase, in the presence and absence of L420A. This work revealed that mutations L420A and G64S operate independently of each other by affecting different steps in the catalytic cycle with G64S increases in fidelity predominately from monitoring nucleosugar positioning while L420A affects nucleobase positioning and polymerase grip on the product RNA strand.Item Open Access Phosphorus ligand-coupling reactions for the functionalization of pyridine and other azines(Colorado State University. Libraries, 2021) Nottingham, Kyle G., author; McNally, Andrew, advisor; Paton, Robert, committee member; Henry, Chuck, committee member; Cohen, Robert, committee memberPyridines and related azines are ubiquitous in pharmaceuticals, agrochemicals, and materials. The discovery and development of new purpose-built molecules is contingent on our ability to modify these motifs. Described herein are the development of methods that selectively functionalize pyridine and diazine scaffolds through phosphorus ligand-coupling. Novel phosphine reagents were designed and leveraged to construct C–C, C–O, and C–N bonds on azines from their C–H precursors. Chapter one introduces the history of phosphorus ligand-coupling and defines the reactivity explored throughout this thesis. Both seminal and contemporary examples of phosphorus ligand-coupling reactions are also discussed to provide context for this work. Chapter two focuses on a method to incorporate fluoroalkyl groups onto azines and pharmaceuticals using phosphorus ligand-coupling. This method offers a complementary alternative to widely used radical addition approaches which often produce regiomeric product mixtures on azines. Chapter three presents the investigation of a phosphorus-mediated alkenylation reaction on pyridines and quinolines. Examination of the reaction of pyridylphosphines with alkyne acceptors uncovered divergent reaction pathways from alkenylphosphonium salts. Mechanistic studies provide an explanation for the origin of selectivity obtained in these reactions. Lastly, chapter four expands upon one of these reaction pathways and describes the development of a method for the direct conversion of pyridines into pyridones and aminopyridines.