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University, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

Azole sind wichtige Chemikalien, die als Fungizide in der Landwirtschaft und der Medizin eingesetzt werden. Auch als Zytostatika in der Humanmedizin finden sie Anwendung. Die fungizide Wirkung beruht auf der Hemmung der Lanosterol-14α-Demethylase (CYP51), die die Demethylierung von Lanosterol zum „Follicular Fluid Meiosis Activating Steroid (FF-MAS)“ katalysiert. Für Pilze ist das später resultierende Ergosterol ein essentieller Bestandteil der Zellmembran. Exponierten Pilzen fehlt Ergosterol was zu einem Zusammenbruch der Zellmembran führt. Säugetiere können Cholesterol, das spätere Produkt der Lanosterol-14α-Demethylierung, das zur Synthese von z.B. Gallensäuren und Sexualhormonen nötig ist, mit der Nahrung aufnehmen. FF-MAS und das resultierende T-MAS (Testis Meiosis Activating Steroids), die direkten Produkte der CYP51 katalysierten Reaktion, wirken als Meiose-aktivierende Steroide auf Ovarien und Hoden und werden nicht mit der Nahrung aufgenommen. Eine Hemmung der CYP51 Aktivität könnte das endokrine System beeinflussen und wird daher als unerwünschte Nebenwirkung der Azole betrachtet. Aromatase (CYP19) katalysiert die Demethylierung von Testosteron zu Östradiol und wird durch Azole gehemmt. Die Verringerung der Östrogenspiegel durch CYP19-Inhibition ist das Wirkprinzip der als Zytostatika genutzten Azole, bei den Fungiziden wird es als unerwünschte Nebenwirkung angesehen. Ein ideales Azol sollte Pilz-CYP51 stark inhibieren, aber sowohl humanes CYP19 wie auch humanes CYP51 sollten durch ein solches Azol nicht inhibiert werden. Ein ideales Azol-Zytostatikum sollte eine starke inhibitorische Potenz gegenüber humanem CYP19 aufweisen, hingegen sollten humanes und Pilz-CYP51 nicht inhibiert werden. Ziel dieser Arbeit war es nun festzustellen: sind Fungizide und Antimykotika starke Inhibitoren von Pilz-CYP51? Zeigen Fungizide und Antimykotika keine Aktivität gegenüber humanem CYP19 und humanem CYP51? Sind Zytostatika starke Inhibitoren von humanem CYP19? Zeigen Zytostatika keine Aktivität gegenüber humanem CYP51 und Pilz-CYP51? Die inhibitorische Potenz von 22 Azolen, aus den drei Anwendungsgebieten, wurden an vier Systemen getestet: i) an humanem CYP19 und einem fluoreszierenden Pseudosubstrat, ii) an CYP19 und Testosteron als Substrat, iii) an humanem CYP51 und iv) Candida albicans CYP51 und Lanosterol als Substrat. Die Produktbildung wurde mittels Hochdruckflüssigkeitschromatographie gekoppelter Tandem-Massenspektrometrie nach Photosprayionisation gemessen. Das humane CYP51 wurde von „BD Gentest Cooperation“ zur Verfügung gestellt. Ein katalytisch aktiver Enzymkomplex bestehend aus der Lanosterol-14α-Demethylase von Candida albicans und der Oxidoreduktase von Candida tropicalis, wurde im Baculovirussystem exprimiert. Ein Vergleich der inhibitorischen Wirkstärke der Substanzen auf menschliches CYP19 und CYP51 und Pilz-CYP51 zeigt, dass einige Azole das erwünschte Bild zeigen. Dazu gehören die beiden Zytostatika Fadrozol und Letrozol, sowie Fluconazol und Itraconazol, zwei Antimykotika aus der Humanmedizin, und einige Fungizide z.B. Cyproconazol und Hexaconazol. Ein unerwünschtes Bild zeigen z.B. Prochloraz, Bifonazol, Ketoconazol und Miconazol. Sieben Azole weisen ein gemischtes Bild an inhibitorischen Wirkstärken auf. Um einen modellartigen Eindruck der Rückstände von Azolen in Lebensmitteln zu erhalten, wurde eine auf LC-ESI-MS/MS basierende Rückstandsanalytik für Azole im Wein entwickelt. Alle gefunden Rückstände lagen unterhalb der behördlich festgelegten Rückstandshöchstmengen. Um die inhibitorische Wirkung der Azole auf die verschiedenen Enzymsysteme in einem größeren Zusammenhang zu bringen, wurden die IC50 Werte mit Expositionsdaten von Bauern, maximalen Plasmaspiegeln in Patienten nach der Einnahme von Antimykotika und mit Expositionsgrenzwerten für die Langzeitaufnahme von Pflanzenschutzmittelrückständen („Acceptable Daily Intake Levels“, ADI) verglichen. Basierend auf den dargestellten Ergebnissen können folgende Schlussfolgerungen gezogen werden. Das Risiko für landwirtschaftliche Arbeiter durch Exposition gegenüber Azolfungiziden kann im Bezug auf menschliches CYP19 und CYP51 als vernachlässigbar eingestuft werden, wenn die entsprechenden Sicherheitsvorkehrungen getroffen werden. Im medizinischen Bereich muss grundsätzlich der Einsatz von Bifonazol, Miconazol und Ketoconazol mit Blick auf die hohe inhibitorische Potenz gegenüber menschlichem CYP19 und 51 kritisch betrachtet werden. Unter der Annahme, dass die ADI Werte eingehalten werden, stellen Rückstände auf Lebensmitteln in Bezug auf die genannten Enzymsysteme keine Bedrohung für den Verbraucher da. Die Inhibition von CYP19 muss als Störung des Hormonsystems angesehen werden. Die Bedeutung von FF-MAS und T-MAS im endokrinen System muss noch abschließend geklärt werden und damit auch die Frage, wie viel Bedeutung der Inhibition von menschlichem CYP51 beigemessen werden muss. / Azoles are important chemicals used as antifungal agents in agriculture and human medicine, but also as cytostatic drugs in tumour chemotherapy. Antifungal activities are based on inhibition of lanosterol-14α-demethylase (CYP51). CYP51 catalyses the oxidative removal of the methyl group # 32 of lanosterol to produce follicular fluid meiosis activating steroid (FF-MAS). For fungi the later resulting ergosterol is an essential compound of the cell membrane. Exposed fungi lack ergosterol, which leads to a collapse of the cell membrane. In mammals cholesterol, the downstream product of lanosterol-14α-demethylation necessary for the synthesis of bile acids, mineral corticoids, glucocorticoids and sex steroids, can be supplemented with food intake. However FF-MAS and the resulting T-MAS (testis meiosis activating steroids), the direct products of the CYP51 reaction, act as meiosis-activating steroids on ovaries and testes and are not supplemented with food intake. Inhibition of CYP51 in humans may therefore affect the endocrine system and is an unwanted side effect of azoles. Aromatase (CYP19) catalyses the demethylation of testosterone to estradiol and is inhibited by azoles. Reduction of estrogen levels by CYP19 inhibition is the working principle of cytostatic drugs used in breast cancer therapy but is considered an unwanted side effect for azoles used to treat fungal infections. A favourable fungicide or antifungal drug should be a strong inhibitor of fungal CYP51. In contrast human CYP51 and human CYP19 should not be inhibited by an azole fungicide or antifungal agent. The favourable cytostatic drug should show a high potency towards human CYP19. Neither human CYP51 nor fungal CYP51 should be inhibited by a cytostatic drug. The aim of this work was to assess: are fungicides and antifungal drugs strong inhibitors of fungal CYP51? In return do they not inhibit human CYP51 and human CYP19? Do cytostatic drugs strongly inhibit human CYP19? And in return do they not inhibit human CYP51 or fungal CYP51? Inhibitory potencies of 22 azole compounds used for the three purposes were tested in four inhibition assays: i) on commercially available human CYP19 utilising a fluorescent pseudo substrate dibenzylfluorescein (DBF) ii) on CYP19 utilising testosterone as substrate iii) on human CYP51 and iv) Candida albicans CYP51 utilising lanosterol as substrate. Product formation was measured by liquid chromatography – tandem mass spectrometry utilising photospray ionisation (APPI). A functional human CYP51 was available from BD Gentest Cooperation. A functional enzyme complex comprising of the Candida albicans lanosterol-14α-demethylase and the Candida tropicalis oxidoreductase was expressed in the baculovirus system. When comparing inhibitory potencies on CYP19, human CYP51 and Candida albicans CYP51 a number of agents show desirable patterns of inhibition e.g. the two cytostatic drugs, or two antifungal agents used in human medicine, fluconazole and itraconazole, and a wide variety of the fungicides, e.g. cyproconazole and hexaconazole. Undesirable patterns of inhibition were exhibited by a number of compounds, e.g. prochloraz, bifonazole, ketoconazole and miconazole. Seven compounds show a more complex picture of inhibitory potencies though. To get a picture of residue levels of azoles in food in a model case an LC-ESI-MS/MS method was developed for the determination of azole compounds in wine. All residues were below the maximum residue levels set by authorities. To classify the inhibitory potencies on the different enzyme systems IC50 values obtained were compared to exposure levels measured in farmers, maximum plasma concentrations in humans reported after exposure to antifungal drugs and to acceptable daily intake levels set by authorities. Based on the findings presented, the following conclusions can be drawn. The risk for agricultural workers set by exposure to azole fungicides with respect to human CYP51 and CYP19 can be regarded as negligible when safety measures are adhered to. As a matter of principle however, the usage of bifonazole, miconazole and ketoconazole has to be viewed with caution in respect to the high level of inhibition of human CYP51 and/or CYP19. Under the assumption that the acceptable daily intake amounts set by authorities for azole compounds are not exceeded the residues do not pose a threat to consumer safety judged by our findings. Inhibition of CYP19 with the consequence of a reduction of estradiol levels has to be regarded as a possible disrupting effect of the hormone balance. The relevance of FF-MAS and T-MAS in the endocrine system however still has to be evaluated completely bringing with it the question of how much importance has to be attached to the inhibition of human CYP51.

Some interesting, biologically active natural products have been found to contain poly-azole fragments within their core. These fragments are linked through the 2- position of one azole and the 4-position of its neighbour. Decarboxylative cross-coupling methodology may provide a route to these desired linked azoles through cross-coupling of azole-4-carboxylic acids with azole-2- halides or with azoles containing no substitution at the 2-position. Investigations into the silver-mediated decarboxylation, and subsequent coupling potential, of thiazole and oxazole-4-carboxylic acids are reported. Methods towards the synthesis of novel chlorinated thiazole and oxazole acids and their precursors are also described. A method to successfully couple these acids to aryl iodides has been developed and the scope of this reaction extended to a variety of functionalised azole-4-carboxylic acids. Attempts to extend this methodology and combine the decarboxylative coupling with CH activation of a second azole are also described.

Aspergillus fumigatus is a globally present pathogen capable of inflicting debilitating and life-threatening opportunistic infections in individuals, primarily those who are immunocompromised. Diagnosing A. fumigatus infections is often difficult, leading to a delay in treatment which can greatly impact patient outcomes. Furthermore, our lessening of antifungal development combined with increasing resistance generates a feasible scenario where only last resort options are viable. This has prompted the World Health Organization (WHO) to declare this pathogen a "critical priority" due to increased resistance and rising mortality rates. Azoles are utilized as primary treatment options for Aspergillus fumigatus infections such as voriconazole (VRC), itraconazole (ITC), and posaconazole (POS) with a reserve of Amphotericin B (AmB). In the past two decades, the emergence of resistance to azoles has contributed to a 90% mortality rate in resistant cases globally. In this report, we investigated the MedChem Express (MCE) Drug Repurposing Compound Library (4,226 compounds) in conjunction with itraconazole at 0.06 µg/mL against A. fumigatus CDC #738. After the initial screening, we identified compounds known to be antifungals or antiseptics and deselected them. The remaining thirty selected compounds were evaluated through published literature and clinical trial data to determine those candidates with favorable characteristics/properties. Criteria for candidate selection consisted of evaluating the compounds; plasma concentration peak, the time to reach peak, protein binding, oral availability, and drug class. Six candidates were ranked the highest of the previous round –surprisingly 50% of those compounds were HIV drugs, cobicistat, elvitegravir, lopinavir. The remaining three selected compounds are penfluridol, rilapladib, and rolapitant. The combination of itraconazole (ITC), posaconazole (POS), and voriconazole (VRC), with the identified compounds demonstrated promising amounts of synergy, in resistant and susceptible isolates. Further investigation revealed novel properties of ITC and POS when in combination with our compounds of interest. Rilapladib was able to reverse POS, ITC, and VRC resistant strain(s) to a sensitive profile. Growth kinetic assays demonstrate potent anti-germination properties not seen before in the sub-inhibitory doses of azoles. This work demonstrates that high-throughput screening as a viable technique to identify robust antifungal synergizers, allowing for tenable translation to a clinical setting. / Master of Science / Aspergillus fumigatus is a worldwide fungal organism capable of causing disease, particularly in immunocompromised individuals. Infections primarily occur when individuals inhale spores that can remain dormant until the person's immune system is weakened, via disease, cancer, or prescribed drug for surgery. When the immune system is weakened, the spores are more effective at lung colonization. Aspergillus fumigatus infections can be combatted with voriconazole (VRC), itraconazole (ITC), or posaconazole (POS). However, in the past two decades, some fungi have started to develop resistance to azoles, necessitating the use of amphotericin B (AmB), a highly intolerable and final treatment option. In this report, we challenged A. fumigatus CDC isolate #738 with compounds from the MedChem Express (MCE) Drug Repurposing Compound Library (4,226 compounds) with itraconazole at 0.06 µg/mL, an ineffective concentration. We selected combinations and compounds that negated 90% of fungal growth those combinations that contained a known antifungal or antiseptic agent; once identified we deselected any known antifungal or antiseptic agents. The remaining selected compounds were evaluated for favorable drug properties, by reviewing published literature and clinical reports to determine those candidates with favorable characteristics/properties. The combination of ITC, POS, and VRC with the identified compounds demonstrated antifungal enhancement in resistant and susceptible isolates. We observed the reversal of resistance to POS, ITC, and VRC in several isolates when the drug combinations were applied. This demonstrates the importance of evaluating approved and under current review drugs to identify novel properties to aid our dwindling number of effective antifungals. This study provides promising combinational therapies for further evaluation in combating resistant A. fumigatus infections.

Les flavohémoglobines (FlavoHbs) sont des protéines fixant l’oxygène qui possèdent un domaine globine N-terminal lié de manière covalente à un domaine C-terminal réductase contenant une flavine adénine dinucléotide (FAD) et un site de fixation du nicotinamide adénine dinucléotide (phosphate) (NAD(P)H). Ces protéines que l’on retrouve exclusivement chez les microorganismes possèdent une action NO dioxygénase et interviennent donc dans la défense des microorganismes contre les dommages générés par le NO. De par ce rôle essentiel de défense microbienne, les flavoHbs sont considérés comme des cibles attractives des antibiotiques. En particulier, les dérivés azolés ont montré la capacité d’inhiber la fonction NO dioxygénase des flavoHbs par un mécanisme inconnu. Afin de mieux comprendre le mode d’action de ces antibiotiques, nous avons entrepris une étude structurale, enzymatique et spectroscopique sur trois flavoHbs d’intérêt. Les structures tridimensionnelles de la flavoHb de R. eutropha (FHP) en complexe avec le miconazole, l’éconazole et le kétoconazole ainsi que celle de S. cerevisae (YHB) seule et en complexe avec l’éconazole ont été obtenues à des résolutions satisfaisantes permettant de décrire précisément les interactions entre la protéine et les inhibiteurs. Les structures ont révélé des réarrangements conformationnels importants selon la nature chimique de l’inhibiteur et la présence d’acides gras dans la poche de l’hème. Afin de comprendre le rôle fonctionnel des acides gras dans le cycle catalytique de l’enzyme, la structure tridimensionnelle de la FHP en complexe avec l’acide linolénique a été obtenue et des analyses enzymatiques et spectroscopiques ont montré l’importance des acides gras dans la modulation de l’activité de la protéine. Parallèlement, des études sur la FHP, la YHB et la flavoHb de S. aureus (Shb) ont permis de mieux appréhender le rôle des inhibiteurs dans le processus de transfert d’électrons au sein de la protéine. / Flavohemoglobins (FlavoHbs) are oxygen binding proteins which consist of a heme-globin domain fused with a ferredoxin reductase –like FAD and NAD-binding domain. FlavoHbs have been identified exclusively in microorganisms where they play a key role in defence against NO damages by using their NO dioxygenase activity. These proteins are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors by a mechanism that remains elusive. In order to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHPm), econazole (FHPe), and ketoconazole (FHPk) as well as the X- ray structure of S. cerevisae flavoHb in both ligand-free and econazole-bound forms. We describe the interactions between the protein matrix and the inhibitors in a comparative manner and how the bulky structures of the azole inhibitors dictate the profile of the hemebinding pocket and vice versa in flavoHbs.Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHPe and FHPk and dictates the state of the protein. To go further in the compréhension of the fatty acid function in the flavoHbs, we determined the three dimensionnal structure of FHP in complex with linolenic acid. Spectroscopic and enzymatic analyzis confirmed the important role of fatty acids in enhancing the protein activity. We also made studies to understand how azoles modulate the electron transfer process in the flavoHbs.

Numerous contaminants are present in mixtures in the aquatic environment. Among these are the azoles, a group of chemicals that includes both pharmaceuticals and pesticides. Azole fungicides are designed to inhibit lanosterol 14-demethylase (cytochrome P450 (CYP) 51), while other azoles are intended to inhibit aromatase (CYP19), i.e. the enzyme catalyzing biosynthesis of estrogens. In fish, a variety of CYP enzymes are involved in biotransformation of waterborne contaminants, and in metabolism of endogenous compounds including steroidal hormones. The induction of CYP1A protein and 7-ethoxyresorufin O-deethylase (EROD) activity are common biomarkers for exposure to aryl hydrocarbon receptor (AhR) agonists in fish. We developed an assay to measure inhibition of CYP1A activity (EROD) in three-spined stickleback and rainbow trout gill tissue ex vivo. Several azole fungicides were found to be potent inhibitors of CYP1A activity. A wastewater effluent containing high concentrations of pharmaceuticals was also shown to inhibit CYP1A activity. Further, several azoles inhibited CYP19 activity in rainbow trout brain microsomes in vitro. Azole mixtures reduced both CYP1A and CYP19 activity monotonically and in an additive way. Given the additive action of the azoles, studies to determine adverse effects of azole mixtures on CYP-regulated physiological functions in fish are needed. Induction of EROD and of gene expression of CYP1 in several organs was observed in an in vivo exposure with the same effluent shown to inhibit EROD. This finding could imply that there was a mixture of AhR agonists and CYP1A inhibitors in the effluent. Finally, wastewater treatment technologies were evaluated using biomarker responses in rainbow trout exposed to effluents of different treatments. The results from chemical analysis together with the biomarker results show that ozone and granulated active carbon treatment removed most pharmaceuticals, as well as AhR agonists and other chemicals present in the regular effluent. This part of the thesis demonstrates that biomarkers in fish such as induction of CYP1 gene expression are applicable to evaluate the efficiency of different treatment technologies for wastewater.

Several Fungal species have the potential to cause a broad spectrum of diseases in humans, ranging from mild superficial to disseminated invasive infections that involve the bloodstream and vital organs. Invasive fungal infections are severe, life-threatening diseases that result in the deaths of 1.5 million patients each year. The most common fungal species responsible for the majority of invasive fungal infections include Candida, Cryptococcus, and Aspergillus. The current treatment options for invasive fungal infections are restricted to three classes of antifungals: Azoles, polyenes, and echinocandins. The emergence of new fungal species, especially C. auris, marked by high resistance profiles and increased mortality rates (30-60%), has further exacerbated the limitations in its therapeutic options. This emphasizes the urgent need for effective alternatives to combat these deadly pathogens. C. auris isolates exhibited high resistance capability especially against azole (fluconazole) and polyene (amphotericin B) antifungals. Here, we utilized the combinatorial strategy to screen ~3400 FDA-approved drugs and clinical compounds to identify hits that can enhance/restore the antifungal activity of azoles and amphotericin B against resistant C. auris. The HIV protease inhibitors (lopinavir and ritonavir) were identified as potent enhancers to the antifungal activity of azole drugs (fluconazole, voriconazole and itraconazole). We confirmed that lopinavir and ritonavir have the capability to interfere with fungal efflux pump machinery. The in vivo efficacy of the combination of azole antifungals and HIV protease inhibitors was also evaluated to discover the best combination of itraconazole, lopinavir and ritonavir. Three drugs (lansoprazole, rolapitant and idebenone) were identified to effectively enhance the antifungal effects of amphotericin B and overcome its resistance in C. auris. Furthermore, the synergistic interactions of these combinations were applied on other medically important Candida, Cryptococcus, and Aspergillus species. In a comprehensive mechanistic study, we discovered that lansoprazole interferes with an essential target in the fungal mitochondrial cytochrome system, cytochrome bc1. This interference induces oxidative stress in fungal cells and subsequently enhances the antifungal activity of amphotericin B. For rolapitant, a transcriptomic analysis along with ATP luminescence assays confirmed that rolapitant at sub-inhibitory concentrations significantly interferes with ATP production in C. auris. For idebenone, checkerboard assays confirmed the synergistic interactions between amphotericin B and idebenone against a diversity of medically important fungal species. This combination exhibited a rapid fungicidal activity within 4 hours. Additionally, the cytotoxicity of this combination was assessed in a cell line model of kidney cells. Based on the potent in vitro and in vivo synergistic relationships observed for the identified combinations, it can be concluded that our approach offers a new hope to restore the antifungal activity of the existing antifungal drugs, even against resistant fungal infections. Additionally, it provides valuable insights into identifying novel targets to overcome resistance in multidrug-resistant fungal pathogens. / Doctor of Philosophy / Fungi comprise a diverse group of organisms that interact with humans in many good and bad aspects. Candida auris, a recently identified fungus, poses a significant threat to patients with weak immune systems. Infections with C. auris can be associated with mortality rates of up to 60%. Notably, this fungus is characterized by its powerful spreading capability and displays extraordinary resistance to antifungal agents, rendering many existing antifungal drugs ineffective. As a result, there is an unmet need to find efficient treatments for such deadly fungal infections. In this study, several drugs were identified with the potential to restore the activity of traditional antifungal drugs. The study identified four promising drugs (lopinavir, lansoprazole, rolapitant, and idebenone) with the potential to enhance the activity of the antifungal drugs against C. auris. lopinavir showed great potential to enhance the activity of azole antifungals, including fluconazole, voriconazole, and itraconazole. Furthermore, three other drugs (lansoprazole, rolapitant, and idebenone) were identified for their potential to enhance the activity of amphotericin B, which is considered a last-line antifungal therapy. We clarified the mechanisms by which these drugs could restore the activity of antifungal agents. Finally, we confirmed the effectiveness of these combinations in animal models, providing valuable insights into their potential for clinical applications. In summary, our research has opened promising avenues to overcome resistance and develop new treatments for hard-to-treat fungal infections.

This research study investigates the effect of micronized copper azole type C (MCA-C) preservative system on the rolling shear (RS) properties of CLT. In the first part of research, bonding performance of CLT panels treated at two retention levels, 0.96 kg/m3 and 2.5 kg/m3, were evaluated. Three structural adhesive systems, melamine formaldehyde (MF), resorcinol formaldehyde (RF) and one-component polyurethane (1C-PUR) were used to assemble visually graded No. 2 2×6 southern yellow pine (SYP) lumber while manufacturing CLT panels. For treated CLT panels, 1C-PUR provides better bonding performance test results. The RS properties of MCA-C treated CLT panels were studied in the second part of the research. The CLT panels were subjected to out-of-plane loading according to the EN 16351 standard. The mean values of RS strength and modulus of treated CLT were 1.89 MPa and 289.4 MPa respectively.