Browsing by Subject "Tuberculosis"

Now showing 1 - 14 of 14
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    Cd4 T Cell Activation And Ox40 Agonist Immunotherapy In Tuberculosis
    (2023-09) Gress, Abigail
    After Mycobacterium tuberculosis (Mtb) infection millions of effector T cells traffic to the lungs, but relatively few find antigen and become activated. We used an antigen receptor reporter mouse (Nur77-GFP) infected with Mtb to distinguish recently activated CD4 T cells from others in the lungs. Recently activated Nur77-GFPHI cells more often expressed protective markers and were enriched for expanded TCR clonotypes. Nur77-GFPHI cells differentially expressed co-stimulatory genes including Tnfrsf4/OX40 and were functionally more protective than Nur77-GFPLO. Nur77-GFPLO cells more often expressed markers of terminal exhaustion, cytotoxicity, and the trafficking receptor S1pr5, associated with vascular localization. A short course of immunotherapy with an agonist monoclonal antibody targeting OX40+ cells during early infection transiently expanded CD4 T cell numbers and shifted their phenotype towards parenchymal protective cells, which decreased the lung bacterial burden and extended host survival, offering an additive benefit to antibiotics. We have newly identified OX40 as a conserved marker of recently activated CD4 T cells at the infection site and a target for immunotherapy in tuberculosis.
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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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    Development of Antibacterial Compounds to Target Drug Resistant Bacteria
    (2021-05) Schultz, John
    Nosocomial infections caused by resistant Gram-positive organisms are on the rise, presumably due to a combination of factors including prolonged hospital exposure, increased use of invasive procedures and pervasive antibiotic therapy. Compounding the problem is the emergence of multidrug-resistant (MDR) Gram-positive bacteria [e.g. methicillin resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae and Enterococcus spp.], which render treatment extremely difficult. As a result, last resort antibiotics (e.g. vancomycin, linezolid and daptomycin) are frequently deployed as treatment for these infections, which have the unintended consequence of selecting resistance to these agents. Although antibiotic stewardship and infection control measures are helpful, newer agents against MDR Gram-positive bacteria are urgently needed. Here we describe our efforts that lead to the identification of 5-aminoquinolone 111 with exceptionally potent Gram-positive activity with MICs ≤ 0.06 µg/mL against numerous clinical MRSA isolates. Preliminary mechanism of action and resistance studies demonstrate the 5-aminoquinolones are bacteriostatic but become cidal with 4-8 times MIC, do not select for resistance, and selectively disrupt bacterial membranes over eukaryotic membranes. While the precise molecular mechanism has not been elucidated, the lead compound is non-toxic displaying a therapeutic index of greater than 1000, is devoid of hemolytic activity and has attractive physicochemical properties (clogP = 3.8, MW = 441) that warrant further investigation of this promising antibacterial scaffold for treatment of Gram-positive infections.Another infectious disease that has a massive burden upon the global society and shares the same concern of drug resistance is Tuberculosis (TB). Mycobacterium tuberculosis (Mtb), the causative infectious agent of TB, contains many essential biosynthetic pathways necessary for the survival and virulence of Mtb, but are absent in humans making these pathways prime candidates for antimicrobial compounds. Chorismate biosynthesis is one such essential pathway that has been exploited as a route to TB chemotherapy. Chorismate is also a metabolic hub towards the biosynthesis of a wide array of aromatic small molecules such as folates, mycobactins, aromatic amino acids, and menaquinone in Mtb. Herein, we describe the synthesis of the epimers of 6-fluoroshikimate and methyl-6-fluoroshikimate, known inhibitors of chorismate-utilizing pathways, and biological evaluation of methyl (6S)-6-fluoroshikimate (125) in Mycobacterium. Initial supplementation studies indicate that methyl (6S)-6-fluoroshikimate may act upon unexpected chorismate-utilizing pathways.
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    Fragment based inhibitor design of Mycobacterium tuberculosis BioA
    (2015-01) Dai, Ran
    7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis (Mtb) is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). We herein report our fragment based inhibitor design of Mtb BioA. Using differential scanning fluorimetry (DSF) fragment screening, the Maybridge Ro3 library of 1000 molecules was screened. Twenty-one compounds giving rise to Tm shifts exceeding ±2°C were then investigated in crystallographic experiments. Six fragments have been co-crystallized with BioA to characterize binding. Each compound has a unique binding mode, and subtle variations in ligand binding site geometry are induced upon binding of different fragment molecules. Binding affinities of the fragments were characterized via isothermal titration calorimetry (ITC). A fragment extension strategy was used to rationally optimize these fragment hits. A commerce based SAR was used and identified 50 compounds containing the core of one of the fragments. These compounds were further screened virtually and experimently by DSF. Four optimized BioA ligands from fragment optimization were validated by X-ray crystallography, including a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5′-phosphate (PLP) cofactor. Binding affinities of these ligands have been characterized by ITC or kinetic assay. The six fragment complex structures were also used for optimization of HTS lead compounds. Six HTS lead compounds were co-crystallized with BioA at high resolution. Design of optimized compounds was by overlapping the fragments and HTS lead binding conformations in the BioA active site. Molecules predicted to have better potency were proposed. Two N-aryl piperazine inhibitors of BioA from HTS optimization were characterized using X-ray crystallography and ITC. One inhibitor that combines features of one HTS lead and one fragment was confirmed with improved binding affinity by ITC.
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    Investigating the RegX3-Dependent Regulation of Protein Release from Mycobacterium tuberculosis
    (2020-07) White, Dylan
    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. As a facultative intracellular pathogen, Mtb is well-adapted to survive within the host and establishes a persistent infection in humans despite a strong immune response. As part of its survival strategy, Mtb releases a wide array of proteins that aid in nutrient acquisition and immune evasion. Broadly, the work presented in this thesis focuses on how an Mtb response regulator, RegX3, regulates protein export by the bacteria, and how export of these proteins impacts Mtb viability. In Chapter 2 we describe that a ∆pstA1 mutant, in which RegX3 is constitutively activated, hyper-secretes a variety of proteins. In previous work, we found that RegX3 induces secretion through the specialized ESX-5 secretion system. We also found hyper-secretion of LpqH, a lipoprotein associated with membrane vesicles (MV) produced by Mtb. Further investigation into this phenotype revealed that ∆pstA1 bacteria release significantly more MV than wild-type (WT) bacteria. We showed that this is a RegX3-dependent phenotype, suggesting a gene(s) within the RegX3 regulon controls MV production. Furthermore, MV release does not depend on ESX-5 activity, so RegX3 induces protein export by at least two different mechanisms. Work in Chapter 3 builds upon the observation that ∆pstA1 bacteria produce more MV. Because this strain constitutively activates RegX3, we leverage it as a tool to identify factors that drive MV release. We hypothesized that the deletion of a specific RegX3-induced gene in the ∆pstA1 background would restore MV production to WT levels, and that the product of this gene would provide information about the physical process of MV release through the Mtb cell wall. We demonstrate that only the deletion of lppF or whiB3 reduce MV production. While WhiB3 was previously reported to transcriptionally regulate the production of cell wall lipids, we found no evidence to support this regulatory mechanism in our ∆whiB3 strains. We conclude that no single gene drives the increased MV production by the ∆pstA1 strain; activation of WhiB3 is responsible for a portion of the phenotype and additional RegX3-dependent factors also contribute. Chapter 4 focuses on the biological functions of ESX-5 activity, specifically to determine how ESX-5 affects Mtb growth and what proteins are secreted by the system. Creating strains harboring tetracycline-repressible copies of ESX-5 components allowed us to circumvent ESX-5 essentiality while still controlling its activity. Using these strains, we found that Mtb requires ESX-5 activity for growth when either glycerol or glucose was the sole carbon source. Additionally, ESX-5 activity was required for optimal growth and survival in both resting and interferon-gamma activated murine macrophages in vitro. It remains to be investigated whether this intracellular survival is connected to ESX-5-dependent nutrient acquisition. These strains also allowed us to perform the first full proteomic analysis to identify protein substrates secreted via ESX-5. Our results uncovered that ESX-5 may play a role in the release of a wider range of proteins than was previously appreciated. We report that release of proteins associated with the cell membrane/cell wall and also proteins secreted via other systems rely on ESX-5 activity to reach the culture filtrate. Taken together, the results presented in Chapter 4 uncover new roles of the ESX-5 system and highlight the importance of this system to Mtb viability.
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    Latent Tuberculosis and Risks of isoniazid therapy
    (2008-02-05) Quinn, Kevin
    Liver damage from isoniazid therapy for latent tuberculosis occurs in 5-10 people per 1000 patients. The risk is greatest in those over 35 years of age.
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    The mechanistic basis of susceptibility and resistance to the antitubercular drug para-aminosalicylic acid
    (2019-05) Kordus, Shannon
    Tuberculosis (TB) is responsible for the deaths of 1.6 million people worldwide each year and is the leading cause of death by the pathogen Mycobacterium tuberculosis. The treatment regimen for M. tuberculosis involves lengthy, intensive drug therapy that causes severe side-effects. The antimicrobial para-aminosalicylic acid (PAS) is used to treat drug-resistant M. tuberculosis infections. Despite the use of PAS to treat M. tuberculosis for over 70 years, the biochemical mechanisms which govern PAS susceptibility and resistance in M. tuberculosis are incomplete. The focus of this dissertation is to determine these mechanisms and can be summarized into three interrelated studies: 1) The mechanism of action of PAS on the M. tuberculosis folate metabolic pathway; 2) Understanding the mechanisms of PAS resistance; and 3) Examining the drug-drug interactions between PAS and other anti-folate drugs that are used to treat opportunistic infections in patients with HIV-M. tuberculosis co-infections. Chapter 2 shows that PAS is a pro-drug and is converted, via the M. tuberculosis folate biosynthetic pathway, to hydroxy-dihydrofolate. The folate biosynthesis pathway is an essential metabolic pathway used maintain the production of DNA, RNA, and proteins. Results showed that hydroxy-dihydrofolate acted as a potent inhibitor for dihydrofolate reductase and confirm the biochemical mode of action of PAS. Although PAS was originally found to be effective at inhibiting M. tuberculosis and showed no activity against other bacterial species. PAS activity was tested against other bacterial pathogens. While M. tuberculosis was extraordinarily sensitive to PAS, other bacteria resisted PAS-mediated killing. Chapter 2 found these bacterial species could utilize PAS as a fully functional folate analog into one-carbon metabolism. The folate biosynthesis precursor, para-aminobenzoic acid (PABA), is a known antagonist of antifolates, namely sulfonamides. Since PAS was shown to act similarly to PABA in folate biosynthesis, PAS-sulfonamide interactions were tested against a panel of bacterial pathogens. PAS could antagonize sulfonamides in all of these organisms. HIV-infected individuals are given numerous drugs to both treat the HIV infection and prophylactically treat and prevent opportunistic infections. The most commonly prescribed prophylactic drugs are the sulfonamides. These findings strongly support that PAS and sulfonamides should cease to be used in combination in individuals with HIV-M. tuberculosis co-infections. Many patients discontinue treatment of PAS because of the side effects, namely, gastrointestinal distress. Bacteria must make their own folates and many bacteria in the human colon excrete folate to supply human enterocytes with folates. All rapidly dividing human cells require folates for the synthesis of new DNA, RNA, and proteins. All human cells contain folate receptors which recognize the metabolites dihydrofolate or folate. Purified human dihydrofolate reductase enzyme and could use hydroxy-dihydrofolate but much less effectively than the native substrate. Since enterocytes require a large amount of folates for rapid cell growth, enterocytes using hydroxy-dihydrofolate as a source of folates may not be able to grow as fast leading to severe gastrointestinal distress. Indeed, hydroxy-dihydrofolate, not PAS, was cytotoxic to enterocytes and hepatocytes. These findings will allow us to ultimately to design better ways to administer PAS to prevent patients from discontinuing PAS treatment. In Chapter 3, we hypothesized mice could be co-treated with sulfonamides to prevent PAS bioactivation and resulting PAS toxicity. Surprisingly, sulfonamides antagonized the anti-mycobacterial action of PAS in mice, resulting in the unrestricted growth of M. tuberculosis in the lungs and dissemination of M. tuberculosis into the liver and spleen. Taken together, these data indicate that combining PAS with sulfonamide in the clinic would not be useful, could be detrimental to patient outcome, and further highlights the need for mechanistic studies of drug-drug interactions. . Chapter 4 established that PAS resistance in M. tuberculosis primarily mapped to the folate biosynthetic pathway. The most prevalent mutations mapped to thyA and folC, a thymidylate synthase and dihydrofolate synthase, respectively. We hypothesized that folC mediated resistance occurred through an increase in PABA biosynthesis. Indeed PABA biosynthesis genes were upregulated in folC resistant mutants but not in thyA resistant mutants. The folC mutants had restored susceptibility to PAS when PABA biosynthesis was disrupted. Furthermore, it was found that disruptions in PABA biosynthesis were bactericidal in M. tuberculosis. These data represent a novel intrinsic mechanism of resistance to PAS and highlights a novel drug target in M. tuberculosis. This dissertation is the first to determine that PAS selectively inhibits M. tuberculosis dihydrofolate reductase enzyme and subsequently, the folate biosynthetic pathway. The work presented in this dissertation found that using PAS and sulfonamides together prevented both drugs from working correctly in M. tuberculosis and in other bacterial pathogens. The results generated from this dissertation will be used to inform the current clinical practices in combination therapy and foster a paradigm shift in the treatment regimen administered to HIV-M. tuberculosis co-infected patients, leading to decreased mortality rates among this population.
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    PPD testing is a valid screening tool for tuberculosis infection in individuals vaccinated with BCG
    (2010-07-29) Lo, Melanie
    Many immigrants in the Twin Cities area come from areas where TB is endemic, and BCG vaccination in infancy is a standard part of TB control efforts. PPD testing is a widely used screening tool in the US, but concern for cross-reactivity in BCG-vaccinated patients may confuse results. This study shows that PPD testing may be used to screen for tuberculosis in adults that were vaccinated with BCG vaccination during infancy. A cutoff induration of 15mm should be used to define a positive PPD test regardless of BCG vaccination status.
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    Pre-clinical drug development of 9-aminoacridines for malignant glioma and meropenem prodrugs for drug-resistant tuberculosis
    (2012-08) Teitelbaum, Aaron M.
    The pre-clinical drug development of investigational molecules with encouraging in vitro efficacy require drug metabolism and pharmacokinetic experiments that ultimately characterize "what the body can do to the drug." In chapter one of this thesis, the absorption, distribution, and metabolic properties of a series of 9-amnoacridines (Acridine 1 - 4) with potent anti-proliferative activity are described by investigating their metabolic stability, cellular accumulation in MDCK cells with or without transfected efflux transporters (PgP or BCRP), plasma protein binding, and tissue accumulation in mouse pharmacokinetic studies. Additionally, acridine 2 was chosen as the lead candidate and evaluated in a mouse orthotopic glioblastoma model. Metabolic stability experiments in pooled human liver microsomes indicated slow rates of oxidation (apparent t1/2 from 2.2 - 4.1 hours) and negligible glucuronidation. In addition, acridine 1, 2, and 4 accumulated in MDCK-WT cells with values 2.7, 2.2, and 4.3 times greater than propranolol. The accumulation of acridine 3 was equal to that of propranolol. In accumulation experiments with MDCK-MDR cells, it was discovered that acridine 1 and 3 were moderate substrates for Pgp whereas acridine 2 and 4 were not substrates. Accumulation experiments with MDCK cells overexpressing BCRP identified acridine 1, 3, and 4 as substrates for BCRP, but not 2. Interestingly, it was discovered that the 9-aminoacridines were substrates for the organic cation transporter (OCT). A mouse pharmacokinetic study following a 60 mg/kg oral dose with Acridine 1 and 2 demonstrated low penetration into the brain (Cmax = 0.25 uM and 0.6 uM), but high uptake in kidney (Cmax = 30 uM and 300 uM) and liver (Cmax = 125 uM and 225 uM) relative to total peak concentrations in plasma (Cmax = 2.25 uM and 20 uM). Subsequently, an intravenous pharmacokinetic study with Acridine 2 following a 15 mg/kg dose produced peak concentrations in the brain (1.7 uM), kidney (212 uM), and liver (78 uM) at 2.0 hours relative to a 2.0 uM peak concentration in the plasma. Acridine 2 bioavailability was 83.8%. Acridine 2 significantly increased the median survival of mice in an orthotopic glioblastoma model suggesting compounds in this series may offer new strategies for the design of chemotherapeutics for treating brain cancers with high oral bioavailability and improved efficacy. Chapter 2 of this thesis describes the synthesis and evaluation of meropenem prodrugs for extensively drug-resistant tuberculosis (XDR-TB). Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of bacterial infectious disease mortality worldwide. Due to the emergence of multi- and extensively drug-resistant strains of TB, new chemical agents are desperately needed. Although B-lactams are the most widely used group of antibiotics, they have never been systematically utilized in TB therapy due to their poor penetration of the mycobacterial cell wall as well as their inactivation by the chromosomally encoded B-lactamase (BlaC). In 2009, Hugonnet et al. shattered this long held dogma demonstrating that meropenem and clavulanate were highly effective against (XDR-TB) strains in vitro. Due to meropenem's short half-life (1 hr) and lack of oral bioavailability, the synthesis and evaluation of meropenem prodrugs as potential therapeutics for the treatment of TB are described. The initial approach to improving the oral bioavailability of meropenem was to synthesize the isopropoxycarbonyloxymethyl (proxetil) ester of meropenem. Second generation prodrugs with more lipophilic promoieties (benzosuberyl, tetralyl, and indanyloxycarbonyloxymethyl) were subsequently synthesized as well as a simple benzyl ester derivative. The aqueous stability of prodrugs at biologically relevant pH 1.2 (stomach), 6.0 (intestinal), and 7.4 (blood) is reported in terms of prodrug half-life or percent remaining after a 2-hour incubation at 37 °C. The most stable prodrug at pH 7.4 and 6.0 was the proxetil ester of meropenem (6 - 11% degradation over 2 hours); however, the prodrugs containing the 1-benzosuberyl, 1-tetralyl, and 1-indanyloxycarbonyloxymethyl promoieties were all unstable in aqueous solution at pH 7.4 (half-lives of 139, 5.8 and < 1 min, respectively) and resulted in the formation of a racemic alcohol indicative of a SN1 solvolysis mechanism. The prodrugs containing the 2-benzosuberyl, 2-tetralyl, and 2-indanyloxycarbonyloxymethyl promoieties were significantly more stable at physiological pH 7.4 and intestinal pH 6.0 (20 - 30% degradation in 2 hours) as a result of carbonate attachment at the 2-poistion and not the benzylic position. The plasma stability of the most aqueous stable prodrugs was very short (1 - 6 min) implying a rapid release of the parent compounds. Interestingly, the simple benzyl ester derivative had the longest stability (8 min) in plasma compared to all the other prodrugs. Experiments to determine the relative bioavailability of meropenem were conducted with the proxetil ester of meropenem as well as the 1-(S), 1-(R), and racemic 2- benzosuberyloxycarbonyloxymethyl prodrugs in jugular vein catheterized guinea pigs. The bioavailabilities were calculated to be 1.9%, 6.2%, 4.4%, and 5.9%, respectively.
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    Robert Koch – From Obscurity to Glory to Fiasco
    (Journal of Opinions, Ideas & Essays (JOIE), 2011-10) Dworkin, Martin
    Robert Koch went from an obscure country physician to be the discoverer of the etiology of anthrax, the inventor of the technique of pure culture bacteriology and with that to the isolation of the tubercle bacillus and its identification as the etiological agent of tuberculosis. These successes propelled him to world-wide glory. In his search for a cure for tuberculosis, he proposed that tuberculin was such a cure. Unfortunately this turned out to be false, and his continued advocacy was a fiasco. Nevertheless his formulation of the germ theory of disease transformed medicine and led to a remarkable series of successes that clarified the etiology of a large number of infectious diseases.
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    Synthesis and Evaluation of Transvalencin Analogues and Adenylation Inhibitors as Antitubercular Agents and Chemical Probes
    (2013-06) Nelson, Kathryn Marie
    >Mycobacterium tuberculosis (Mtb), the etiological agent of pulmonary tuberculosis, is the leading cause of death due to an infectious disease worldwide. Due to a lack of new drug development, poor fidelity to currently available therapeutics, and repeated exposure to therapeutics, Mtb has become multidrug resistant, extensively drug resistant, and even totally drug resistant in some patients. With nearly 9 million deaths and 1.4 million new cases reported by the World Health Organization (WHO) in 2011, new therapeutics that act by novel mechanisms of action are desperately needed to fight this global health threat. Herein we describe our efforts to develop new antitubercular agents by attacking the bacteria's need for iron. This approach involves the inhibition of the biosynthetic pathway to produce siderophores, small molecule iron chelators responsible for acquiring iron in limiting conditions, such as a human host. A prototypical inhibitor of the initiating enzyme in this pathway, MbtA, had previously been developed by our lab, and was used to develop a small set of analogues for in vivo evaluation. We employed Sprague-Dawley rats to evaluate the oral bioavailability of our compounds, revealing that the pKa of the linker nitrogen of the scaffold had a large effect on compound permeability.In addition, we studied the mechanism of action of our parent inhibitor, Sal-AMS, through the development of a photoaffinity probe to label and pull down proteins for target identification. A probe containing a benzophenone moiety for photo-crosslinking and a small alkyne handle for attachment of an imaging or enrichment tag was successfully synthesized. This probe was successful in identifying the intended enzyme of interest (MbtA) as a binding partner, but did not yield any additional hits, suggesting Sal-AMS is a highly specific inhibitor. We also studied a natural product, transvalencin Z, that had been reported as selective against mycobacteria. This compound was very similar in structure to the mycobactins from Mtb, suggesting that it might interfere with iron acquisition or homeostasis. We successfully synthesized the 4 possible diastereomers of the reported structure in an attempt to define the absolute stereochemistry of the natural product, but were unable to match spectroscopic data to the literature report. We attempted to confirm the true stereochemistry through activity testing, but again found our negative results to be in stark contrast to those reported by the discovery group. Finally, we aided a collaborator in the development and synthesis of a probe against DhbE in Bacillus subtilis. Dr. Jun Yin of the University of Chicago was studying the substrate specificity of adenylation domains, and chose an enzyme highly homologous to our target MbtA. We designed a probe that incorporated a similar inhibitor, DHB-AMS, and a long flexible linker with a biotin attached for Dr. Yin's unique yeast cell display assay. Dr. Yin was able to utilize these probes to successfully identify mutant adenylation enzymes with altered specificity towards nonnative substrates. This technique is an exciting new way to potentially access analogues of natural products through manipulation of the biosynthetic machinery, instead of through the organic chemist. These studies have continued to advance our understanding of a new mechanism of action against Mtb, and have brought us one step closer to a preclinical candidate.
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    Targeting Mycobacterium tuberculosis intrinsic resistance mechanisms to potentiate antitubercular drug action
    (2019-06) Thiede, Joshua
    The work presented herein has focused on understanding the mycobacterial metabolic responses that contribute to intrinsic resistance to the antitubercular drugs p-aminosalicylic acid and pyrazinamide. The interrogation of these responses has led to improved understanding of the underlying mechanisms conferring intrinsic resistance to each drug. Leveraging our observations, we were able to improve the antitubercular action of p-aminosalicylic acid by several orders of magnitude as well as circumvent a known mechanism of resistance. Further, we were able to decipher the long observed, but unknown mechanism responsible for conditional pyrazinamide susceptibility. Finally, we developed a method to drive pyrazinamide susceptibility in M. tuberculosis through co-treatment with peptidoglycan targeting drugs. These studies deepen our understanding of how M. tuberculosis withstands drug treatment and offers novel strategies to improve our ability to combat the disease.
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    Targeting Two Late-Stage Enzymes of the Mycobacterium tuberculosis Biotin Biosynthetic Pathway
    (2018-09) Bockman, Matthew
    Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the leading cause of mortality among infectious diseases worldwide. The rise and propagation of drug-resistant TB remains a global health crisis and has prompted researchers to investigate novel mechanisms of action for the development of antitubercular agents. Chapter 1 discusses the biotin biosynthetic pathway as a target for the development of antibiotics targeting Mtb, providing both chemical and genetic validation evidence of inhibiting this pathway in Mtb infections. This chapter thoroughly examines each enzyme in the biotin biosynthetic pathway by reviewing: the reaction it catalyzes, its mechanism of action, structural and sequence analysis, and catalogue of inhibitors known for each enzyme. The late-stage biotin synthase (BioB) and biotin protein ligase (BPL) proteins are elaborated on and will be the focus of this thesis. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. Chapter 2 reports the synthesis and evaluation of a systematic series of potent nucleoside-based bisubstrate inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KDs ≤ 2 nM. Additionally, this chapter discusses the structural interactions between the inhibitors and MtBPL using the highly-resolved x-ray co-crystal structures. Despite relatively uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MICs) ranging from 0.78 to >100 uM. Cellular accumulation studies showed a nearly ten-fold enhancement in accumulation of a C-2′-a-fluoro analogue over the corresponding C-2′-b-fluoro analogue, consistent with their differential whole-cell activity. The parent compound, Bio-AMS, was also evaluated for its pharmacokinetic (PK) parameters, and although it shows stability toward plasma and liver microsomes, Bio-AMS is rapidly cleared form CD-1 mice. From chapter 2, the potent compound Bio-AMS was shown to possess selective activity against MtBPL. However, Mtb develops spontaneous resistance to Bio-AMS with a frequency of resistance (FOR) of at least 1 x 10-7 by overexpression of Rv3406, a type II sulfatase that enzymatically inactivates Bio-AMS. In an effort to circumvent this resistance mechanism, chapter 3 describes the strategic modification of the Bio-AMS at the 5’-position to prevent enzymatic inactivation. The new analogues retain subnanomolar potency to MtBPL, and the 5′R-C-methyl derivative exhibited identical antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an isogenic Rv3406 overexpression strain (MIC = 1.56 uM). Moreover, this compound was not metabolized by recombinant Rv3406 and resistant mutants to this compound could not be isolated (FOR < 1.4 x 10-10) demonstrating it successfully overcame Rv3406-mediated resistance. The natural product acidomycin, discovered in 1952 and isolated from Streptomyces spp., was originally shown to have selective antibiotic activity against Mtb grown in the absence of biotin, implying it is an antimetabolite of the biotin biosynthetic pathway. Chapter 4 fully investigates the mechanism of action and selectivity of acidomycin. Acidomycin was evaluated against an array of drug susceptible and drug resistant Mtb strains, as well as a panel of gram-positive and gram-negative pathogens, and showed remarkable selectivity to Mtb with MICs ranging from 0.096 – 6.2 uM for the Mtb strains and >100 M for the other microorganisms. Acidomycin was also shown to be a reversible, competitive inhibitor of E. coli biotin synthase (EcBioB), with a Ki of 1.5 uM, and a homology model shows substantial sequence alignment in the mycobacterial enzyme (MtBioB). The selectivity of acidomycin against E. coli versus Mtb is due to differential levels of cellular accumulation, with a 30-fold increase in the amount of acidomycin accumulated in Mtb over E. coli. In vivo, acidomycin was shown to be rapidly eliminated from CD-1 mice, is a half-life of 9.6 min, but exhibited remarkable plasma and microsomal stability. A brief series of acidomycin analogues showed a very tight SAR window for modifications, with the primary amide analogue being the best analogue with an MIC less than two-fold of acidomycin.
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    Tuberculosis and the Mantoux Test: What you need to know
    (2008-02-06) Prabhu, Som
    This patient education brochure informs people about the utility of the Mantoux test as a diagnostic tool for investigation of Tuberculosis. It also informs them about the means of administration of the test and gives general guidelines that explain test results. Additionally it explains the possibility of the BCG vaccine as a possible confound while interpreting test results.