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The alarming rise of antibacterial drug resistance and the dwindling supply of novel antibiotics highlight the need for innovative approaches in combating bacterial infections. Traditionally, antibacterial drug discovery campaigns have largely been conducted in rich media. Such growth conditions are not representative of the host environment and render many metabolic pathways, otherwise needed for survival and infection, dispensable. Such pathways have been overlooked in conventional drug discovery campaigns despite their validity as potential antibacterial targets. The work presented in this thesis focuses on the development and validation of a screening strategy for the identification and mechanism of action determination of novel inhibitors of metabolic pathways in bacteria under nutrient-limited conditions. This screen led to the identification of MAC168425, MAC173979 and MAC13772 as inhibitors that target glycine metabolism, p-aminobenzoic acid biosynthesis and biotin biosynthesis, respectively. Moreover, it established this approach as a general platform that can be applied for different organisms with synthetic or natural product libraries. Additional mechanistic studies of the biotin biosynthesis inhibitor, MAC13772, resulted in solving the crystal structure of BioA in complex with MAC13772. Analysis of the co-structure confirmed our proposed mode of inhibition and provided information for strategies for rational drug design. Investigation of the antibacterial activity of MAC13772 revealed its potency against a number of pathogens. Furthermore, we show how MAC13772 acts synergistically with rifampicin in clearing growing mycobacterial cultures. The potential of this inhibitor as a lead for preclinical pharmacokinetic studies and for antibacterial drug development is discussed. We also discuss our current efforts to develop a metabolomic platform for the characterization of novel antibacterials that can be used in concert with our current approach to chart the metabolic response of bacteria to chemical perturbants and to generate testable hypotheses regarding the mode of action of novel inhibitors of bacterial metabolism. / Thesis / Doctor of Philosophy (PhD)

Anthrax is severe disease caused by the gram-positive Bacillus anthracis that can affect humans with deadly consequences. The disease propagates via the release of bacterial spores that can be naturally found in animals or can be weaponized and intentionally released into the atmosphere in a terrorist attack. Once inhaled, the spores become activated and the anthrax bacterium starts to reproduce and damage healthy macrophages by the release of the anthrax toxin. The anthrax toxin is composed of three virulent factors: (i) anthrax protective antigen (APA), (ii) anthrax lethal factor (ALF), and (iii) anthrax edema factor (AEF) that work in harmony to effectuate the lethality associated with the disease. Out of the two internalized factors, ALF has been identified to play a critical role in cell death. Studies in animals have shown that mice infected with an anthrax strain lacking ALF survive the infection whereas when ALF is present the survivability of the mice is eliminated. Although the current therapy for anthrax is antibiotic treatment, modern medicine faces some critical limitations when combating infections. Antibiotics have proven very efficient in eliminating the bacterial infection but they lack the ability to destroy or inhibit the toxins released by the bacteria. This is a significant problem since ALF can remain active in the body for days after the infection is eliminated with no way of inhibiting its destructive effects. The use of inhibitors of ALF is an attractive method to treat the pathogenesis of anthrax infections. Over the last decade several inhibitors of the enzymatic activity of ALF have been identified. In order to identify inhibitors of ALF a variety of screening approaches such as library screenings, Mass Spectroscopy- based screenings and scaffold-based NMR screening have been used. Results from these iv screening have yielded mainly small molecules that can inhibit ALF in low micromolar to nanomolar concentrations. Yet, although valuable, these results have very little significance with regards to treating ALF in a real-life scenario since pharmaceutical companies are not willing to invest in further developing these inhibitors. Furthermore, the low incidence of inhalation anthrax, the lack of a market for an ALF inhibitor, and the expenses associated with the approval process of the FDA, have hindered the motivation of pharmaceutical companies to pursuit these kind of drugs. Therefore we have screened a small-molecule library of FDA approved drugs and common molecules in order to identify currently approved FDA drugs that can also inhibit ALF (Chapter III). The screening revealed that five molecules: sulindac, fusaric acid, naproxen, ketoprofen and ibuprofen bound to either ALF or APA with sulindac binding both. Additionally, we have developed a nanoparticle-based screening method that assesses molecular interactions by magnetic relaxation changes (Chapter II). Using this assay, we were able to accurately measure the dissociation constants of different interactions between several ligands and macromolecules. Moreover, we have used computational docking studies to predict the binding site of the identified molecules on the ALF or APA (Chapter IV). These studies predicted that two molecules sulindac and fusaric acid could be potential inhibitors of ALF since they bind at the enzymatic pocket. As a result, we tested the inhibitory potential of these molecules as well as that of the metabolic derivatives of sulindac (Chapter V). Results from these studies provided conclusive evidence that fusaric acid and sulindac were both strong inhibitors of ALF. Furthermore, the metabolic derivatives of sulindac, sulindac sulfide and sulindac sulfone v also inhibited ALF. Overall, taking together these results we have discovered the alternate use of a currently used drug for the treatment of ALF pathogenesis.

Alzheimer’s disease (AD) drugs in therapy are limited to acetylcholine esterase inhibitors and memantine. Newly developed drugs against a single target structure have an insufficient effect on symptomatic AD patients. Results: Novel aromatically anellated pyridofuranes have been evaluated for inhibition of AD-relevant protein kinases cdk1, cdk2, gsk-3b and Fyn. Best activities have been found for naphthopyridofuranes with a hydroxyl function as part of the 5-substituent and a hydrogen or halogen substituent in the 8-position. Best results in nanomolar ranges were found for benzopyridofuranes with a 6-hydroxy and a 3-alkoxy substitution or an exclusive 6-alkoxy substituent. Conclusion: First lead compounds were identified inhibiting two to three kinases in nanomolar ranges to be qualified as an innovative approach for AD multitargeting.

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No description available.

Interstrand cross-links (ICLs) are a type of DNA damage that prevents strand separation required for basic cellular processes. ICL-based anti-cancer therapies exploit the cytotoxic consequences of replication and transcription inhibition, however, they are limited by the ability of the cell to repair DNA crosslinks. The challenge of ICL repair involves coordinating multiple DNA repair pathways to remove damage occurring on both strands of DNA. Participation of factors that are both exclusive and essential to crosslink repair suggests a pathway requirement to process unique structures and/or intermediates arising only in ICL repair. SNM1A is a nuclease required for survival of human cells in response to ICL exposure, but the specific function and role of SNM1A remain unclear. Here we show that, in addition to known 5’-3’exonuclease activity, SNM1A possesses single-strand specific endonuclease activity. Furthermore, SNM1A exhibits translesion nuclease activity on crosslinks which deform the helical backbone, but not non-distorting stable ICLs. We report the identification and characterization of nine small molecules inhibitors of SNM1A, isolated from an in vitro high-throughput screen of nearly 4,000 bioactive compounds. Finally, we demonstrate that inhibitors of SNM1A potentiate the cytotoxicity of ICL-inducing agent cisplatin in HeLa cells. The work in this thesis expands the possible roles of SNM1A in ICL repair and lays the groundwork for SNM1A inhibition in ICL sensitization efforts. / Thesis / Doctor of Philosophy (PhD)

Übergangsmetall-Oxo, -Peroxo und -Oxyl-Intermediate gelten als essentielle Zwischenstufen von biochemischen Transformationen, welche die Bildung und Spaltung der Disauerstoff -Bindung involvieren. Biomimetische Untersuchungen der katalytisch aktiven Zentren von Enzymen dienen dem Verständnis der verschiedenen Mechanismen katalytischer Reaktionen in biochemischen Systemen. Weiterhin können ebenjene Studien einzigartige Einblicke in den Einfluss scheinbar geringer Variationen der koordinativen Umgebung in die Produktselektivität, der katalytischen Aktivität und der Mechanismen ablaufender Reaktionen liefern. Die Darstellung und Charakterisiserung dieser kritischen Zwischenstufen ist deshalb ein wertvolles Werkzeug zur Aufklärung und Verständnis mechanistischer Abläufe. Aus den so gewonnen Erkentnissen erwächst das Potential neue und optimierte Katalysatoren unter anderem für die Spaltung und Bildung von Disauerstoff entwickeln zu können. Die in der vorliegenden Dissertation beschriebenen Arbeiten zeigen deutlich den Einfluss scheinbar gerinfügiger Änderungen des Ligandensystems bzw. des Reaktionsmediums auf die Bildung, Stabilisierung und Spaltung der Disauerstoff-Bindung. / Transition metal-oxo, -peroxo, -oxyl intermediates have been proposed as key reactive intermediates in biochemical transformations relevant to dioxygen bond formation and cleavage. Biomimetic studies of relevant enzyme active sites help us to understand the different mechanistic pathways operating in biological systems. Moreover, biomimetic studies also provide us with unique insights into the effect of subtle changes in the coordination environment that can influence the product-selectivity, catalytic efficiency and reaction mechanism. The generation and characterization of these crucial reactive intermediates, therefore, provide a valuable tool for mechanistic understanding and consequently, aid in the development of new and improved catalysts for dioxygen bond formation and cleavage. Therefore, in this thesis, we show how subtle changes in the ligand framework or reaction medium can greatly influence the fate of the dioxygen bond, its formation, stabilization and cleavage.