Browsing by Subject "Nucleosides"

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    Design and Development of Antibiotics and Synthesis of Modified Nucleosides
    (2022-10) Hegde, Pooja
    Tuberculosis (TB) remains a leading cause of infectious disease mortality and morbidity resulting in nearly 1.3 million deaths annually and infecting nearly one-quarter of the population. Chapter 1 discusses the epidemiology, control, and management of TB. Isoniazid (INH) is a cornerstone for the treatment of drug-susceptible TB, yet the quantitative structure-activity relationships for INH are not well documented in the literature. Chapter 2 evaluates a systematic series of INH analogs against contemporary Mycobacterium tuberculosis (Mtb) strains from different lineages and several key species of non-tuberculous mycobacteria (NTM). To assess the specific activity of this series of INH analogs against mycobacteria, we assayed them against a panel of gram-positive and gram-negative bacteria, as well as a number of fungi. Our findings provide an updated analysis of the structure-activity relationship of INH that will serve as a valuable resource for the development of a next generation antitubercular compounds. para-Aminosalicylic acid (PAS), is an important second-line agent for treating drug-resistant Mtb. PAS has a moderate bioavailability and rapid clearance that necessitate high doses in order to facilitate an effective treatment. Consequently, such high doses commonly results in gastrointestinal disturbances (presumably by disruption of gut microbiota and host epithelial cells). Chapter 3 discusses the design, synthesis and evaluation of PAS prodrugs and analogs with improved oral bioavailability, thereby preventing intestinal accumulation as well as undesirable bioactivation by the gut microbiome to cytotoxic folate species. The pivoxyl prodrug and fluorination at the 5- position address the primary limitations of PAS and have the potential to revitalize this second-line TB drug. Pyrazinamide (PZA) is a critical component of the first-line TB treatment regimen because of its sterilizing activity against non-replicating Mtb, but its mechanism of action has remained enigmatic. PZA is a prodrug converted to the active moiety- pyrazinoic acid (POA), by pyrazinamidase, an amidase within the nicotinamide adenine dinucleotide (NAD) salvage pathway encoded by pncA in Mtb. PZA resistance is most commonly induced via loss-of-function mutations within PncA. It has recently been demonstrated that POA induces targeted protein degradation of the enzyme PanD, a crucial component of the coenzyme A biosynthetic pathway, essential in Mtb. Chapter 4 describes the structure activity relationship (SAR) evaluation of various POA analogs. Further development and mechanistic analysis of these analogs may lead to a next generation POA analog for treating TB. The salicylic acid derived small molecule siderophores known as mycobactins are essential for mycobacterial iron acquisition and mycobactin biosynthesis has been biochemically and genetically validated as essential for survival in vivo. Sal-AMS, a modified nucleoside derivative that mimics an intermediate in the mycobactin biosynthetic pathway, is a potent Mtb inhibitor. Chapter 5 explores polyfluorinated salicylic acid derivatives as antimetabolites, designed to antagonize mycobactin synthesis. Enzymatic studies demonstrated that the tri- and tetra- fluorinated salicylic acid analogs were neither substrates nor inhibitors of MbtA, but the di-fluorinated compounds were readily activated by the bifunctional adenylating enzyme MbtA, responsible for processing salicylic acid moieties for synthesis of mycobactins. However further microbiological analysis revealed that the polyfluorinated derivatives have low potential as anti-TB agents and do not appear to operate by inhibiting mycobactin synthesis. Lastly, the chemical synthesis of nucleoside analogs, an important class of compounds with applications as anti-infective, anti-cancer, and diagnostic agents, is extremely challenging, typically requiring linear synthesis, multiple protection deprotection sequences, and stereoselective formation of the critical glycosidic linkage. Nucleoside phosphorylases (NPs) have tremendous biocatalytic potential and aid in the selective modification of nucleosides under green conditions. Chapter 6 focuses on the functional characterization of 15 thermostable purine NPs. Four of these were further selected for a comprehensive analysis of their substrate scope. Next, chemoenzymatic methods for the synthesis of modified nucleosides were extensively studied using various coupled systems, and 12 modified nucleosides were synthesized, isolated, and characterized.
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    Synthesis of Photoresponsive Nucleosides and Their Incorporation into Oligonucleotides: Targeting Androgen Receptor and NF-κB Transcription Factors
    (2016-02) Struntz, Nicholas
    The sequencing of the human genome suggests that transcription factors (TFs) make up one of the largest classes of human proteins, revealing that there are over 2000 genes that code for transcription factors. The pivotal roles of TFs in cell biology become quite apparent when one or more of these regulatory mechanisms becomes mutated or altered. For example, the androgen receptor (AR) transcription factor plays a pivotal role in prostate carcinogenesis and progression. Additionally, the inflammatory response of the NF-κB transcription factor proteins results in the transcription of many genes, which play pivotal roles in carcinogenesis. There are several approaches to modulate and study transcription factor activity and biochemistry. Utilizing cis element DNA decoys to sequester TFs is one approach to directly modulate transcription factors. Introducing these synthetic double-stranded DNA decoys containing TF binding sites into cells effectively sequesters TFs and inhibits their target gene expression. Over the past couple of decades, numerous reports have validated utilizing this approach. For example, a phosphorthioate STAT3 DNA decoy has entered the “first-in-human” Phase 0 clinical trials for the treatment of head and neck squamous cancer. STAT3 expression and cell viability was reduced in the head and neck cancers injected with the decoy compared to the saline control. Combining the spatial and temporal resolution of caging technology with the DNA decoy strategy for the inhibition of transcription factor activity can yield an approach for the very precise ability to photochemically regulate gene expression, which has potential as a therapeutic agent and tool for probing biological pathways. This thesis will focus on efforts to develop several novel DNA-based and small molecule-based probes to investigate the biochemistry of TFs and their signaling pathways. Chapter 2 discusses the synthesis and characterization of caged DNA decoys that target the Androgen Receptor (AR). Caged DNA decoys successfully captured AR in LNCaP lysate when irradiated with light. Chapter 3 introduces the complement to caging technology, which is catch and release DNA decoys (CRDDs). CRDDs capture transcription factors, by binding and sequestering them, and then a pulse of light photochemically destroys the CRDD, permitting release of the TF. Several 7-nitroindole (7-NI, 1.47) nucleobase mimics were incorporated into NF-κB-directed DNA decoys, which still allowed the capture of the p50-p65 NF-κB proteins. Irradiation with 350 nm light drives the release of the p50-p65 NF-κB. The capture and photochemical release of an endogenous transcription factor is demonstrated for the first time. Chapter 4 continues the work of Chapter 3 by developing second-generation nucleobase mimics for use in CRDDs. Addition of molecular recognition properties on a photo-responsive monomer is hypothesized to increase binding affinity to capture endogenous TFs. 8-Nitroguanosine contains this added molecular recognition, is more stable within duplex DNA, and also displayed similar photochemical depurination properties. Chapter 5 outlines work developing photoswitchable nucleobases that transpose their hybridization properties upon photolysis. Chapter 6 highlights work to determine the mechanistic NF-κB inhibitory properties of several Cryptocaryone analogues, which were found to inhibit the NF-κB translocation to the nucleus. Appendix A focuses on the characterization of the enantioselectivity of guanosine monophosphate synthetase (GMPS), a crucial enzyme in nucleotide biosynthesis.