Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Description based on contents viewed Oct. 2, 2008; title from PDF t.p. Includes bibliographical references (p. 34-42).

No description available.

Organophosphates (OPs), such as nerve agents and insecticides, potently inhibit acetylcholinesterase (AChE). Oximes, such as the currently FDA approved oxime 2-PAM, remove the OP from the inhibited enzyme. 2-PAM is effective against select OPs and cannot effectively pass the blood-brain barrier to attenuate OP induced CNS damage. Our laboratory has synthesized a series of substituted phenoxyalkyl pyridinium oximes (Patent number: 9,227,937) that have demonstrated increased survival rates compared to 2-PAM. This research investigated 1) in vitro oxime reactivation of rat, human, and guinea pig serum BChE after inhibition by nerve agent and insecticidal OPs; 2) in vitro determination of reactivation kinetic rate constants for OP inhibited human and rat serum BChE and electric eel AChE after inhibition by a sarin surrogate and paraoxon; 3) intranasal delivery of oximes to reactivate brain AChE in vivo after inhibition by a sarin surrogate. Novel oxime 15 demonstrated significant broad spectrum reactivation of OP-inhibited rat serum BChE while novel oxime 20 demonstrated significant broad spectrum reactivation of OP-inhibited human serum BChE. All tested oximes were poor reactivators of OP-inhibited guinea pig serum BChE. Kinetic analysis of reactivation for NIMP and paraoxon human and rat serum BChE and electric eel AChE demonstrated differences in the second order rate constants. Oxime 20 demonstrated reactivation efficiency for both NIMP and paraoxon inhibited rat and human serum BChE and electric eel AChE more effectively than 2-PAM. Intranasal delivery of either oxime 20 or 2-PAM showed attenuation of NIMP-inhibited brain AChE inhibition in select brain regions and select time points. Oxime 20 demonstrated a larger window of effectiveness but neither oxime attenuated brain AChE inhibition in the hindbrain for any time point or for any brain region at the ten minute time point. These data suggest that reactivation of OP-inhibited BChE may be contributing to the observed increases in survival seen with our oximes. Novel oxime 20 demonstrated reactivation efficacy towards both BChE and AChE inhibited enzyme and a rapid entry into the brain after intranasal delivery. Having an oxime that can be effective in a multitude of ways would be of great value to medical and military personnel.

No description available.

The aim of this research was to determine whether a series of novel pyridinium oximes that were synthesized to incorporate phenoxyalkyl moieties to increase lipophilicity, and thereby increase their likelihood of crossing the blood-brain barrier, could effectively reactivate phosphorylated AChE in vitro. An experimental OP was synthesized as a surrogate for sarin to test the reactivation potential of the oximes, phthalimidyl isopropyl methylphosphonate (PIMP). The reactivation activities of the novel pyridinium oximes on PIMP exposed AChE and structure activity relationships were examined. Differences in reactivation potential in comparison to the widely used 2-PAM were also examined. All the novel oximes tested demonstrated some ability to reactivate inhibited AChE. Percent reactivations varied among the oximes (24%-78%), and the novel oximes were not as effective as 2-PAM or TMB-4, which reactivated 91 and 97%, respectively. The lipophilicity for all oximes was greater than 2-PAM or TMB-4 by 3- to 374- fold. A few of the novel oximes showed combined higher lipophilicity and reactivation potential approaching that of 2-PAM, and therefore suggest some potential efficacy as brain-penetrating oxime reactivators.

To examine the interactions of disease and pollution on amphibian populations around the world, I investigated the effect of infection on contaminant susceptibility in pickerel frog, Rana palustris, larvae. I conducted standard 48-hr toxicity tests to examine effect of trematode parasite, Echinostoma trivolvis, infection (0, 10, or 30 cercaria) on the susceptibility of pickerel frog tadpoles to the widely used organophosphate insecticide malathion. LC50 values ranged from 16.5 – 17.4 mg/L, within the range reported for other amphibian species. I found no differences in susceptibility to malathion among parasite treatments. Nevertheless, this crucial question remains to be tested in other amphibian host-parasite systems. Second, I studied the reverse interaction, the effect of pesticide exposure on susceptibility to parasite infection. I exposed pickerel frog embryos to low doses of malathion, then subjected morphologically normal tadpoles to E. trivolvis later in development. Malathion significantly decreased hatching success and viability rates at concentrations lower than previously documented for anuran embryos. After 7 wk of development in water with no malathion, tadpoles previously exposed to malathion as embryos suffered increased parasite encystment rates compared to controls. My research identifies embryonic development as a sensitive window and the potential for increased susceptibility to infection long after pesticide exposure has ceased. With potential for increased parasite prevalence from eutrophication and climate change, my data underscore the importance of understanding the reciprocal influences of parasites and pesticides in amphibians. / Master of Science

Im Rahmen dieser Arbeit wurden verschiedene Inhibitoren der Acetylcholinesterase (AChE) untersucht, die als potentielle Substanzen zur Behandlung von Morbus Alzheimer eingesetzt werden können. Die Hemmwirkung der einzelnen Substanzen wurde mittels Ellman-Test überprüft. Gemeinsames Strukturmerkmal der Substanzklasse, von der im ersten Teil der Arbeit ausgegangen wurde, war das Grundgerüst des AChE-Reaktivators TMB4 [1,1´-Trimethylen-bis(4-Formyl-Pyridiniumbromid)-Dioxim]. Anhand der biologischen Daten konnte beobachtet werden, dass die Art der Substitution die inhibitorische Aktivität der Verbindungen wesentlich beeinflusst. Am wirksamsten von allen Bispyridinium-Derivaten zeigte sich das 2,6-chlorierte Derivat DUO3 (IC50 = 0.34 μM), gefolgt von monobenzyl-substituiertem UNO3, bismethylsubstituiertem TBM und unsubstituiertem TMB4. Weiterhin wurde der Bindungsmodus der DUO-Substanzen im aktiven Zentrum der AChE untersucht. Die Docking-Studien an Substanzen der DUO-Klasse zeigten ein einheitliches Bindungsmodel, welches folgende Wechselwirkungen be-inhaltet: π-π-„stacking“ zwischen dem Benzylring einer DUO-Substanz und dem Trp84 am Grunde der Bindetasche des Enzyms, face-to-face Wechselwirkung (π-π und Kation-π) zwischen dem Pyridiniumring und Trp334 oder Phe331 der aromatischen Furche. Bei 60% der gedockten Strukturen wurde eine face-to-face-Wechselwirkung an der anionischen peripheren Seite (PAS) der AChE-Tasche festgestellt. Weiterhin wurden neue optimierte Inhibitoren entwickelt. Die Bispyridinium-Struktur der DUO-Derivate wurde um den aus der Furche herausragenden Benzylring gekürzt. Als Leitstrukturen dienten die AChE-aktivsten Substanzen DUO3 (2,6-Cl-Derivat) und DUO12 (2-Cl-Derivat) sowie ein bisphthalimidomethyl-substituiertes TMB4-Derivat (WDUO). Die aktivste Verbindung der Pyridinium-Klasse war die Phthalimid-Phenyl-substituierte Substanz 3c (IC50 = 0.073 μM). Ihre inhibitorische Aktivität gegenüber AChE befand sich im Bereich der des Tacrins (IC50 = 0.044 μM). Sie zeigte eine sehr gute AChE-Selektivität; die BChE hemmte sie um den Faktor 34 schwächer. Im Rahmen der vorliegenden Arbeit wurde nicht nur das rationale Design angewandt, um zu viel versprechenden Kandidaten bezüglich klinischen Einsatzes zu gelangen. Auch das Verfahren der „Random Chemistry“ kam zum Einsatz, um neue und interessante Strukturen zu erzeugen, die eventuell bessere Eigenschaften als die Ausgangssubstanz besitzen. Der Grundgedanke dieses Verfahrens liegt in der Ausnutzung der durch gamma-Strahlen (60Co-Quelle) induzierten Radiolyse des Lösungsmittels, welches seine primären Produkte zur chemischen Reaktion mit dem in ihm gelösten Stoff zur Verfügung stellt. Aus den entstandenen Produkten wurden durch spezifische biologische Testung (Inhibition der AChE) positiv reagierende Komponenten herausselektiert. Die Proben wurden zuerst im Ganzen auf ihre Fähigkeit, die AChE zu hemmen, geprüft. Nach der bioaktivitätsgeleiteten Fraktionierung und Subfraktionierung mittels HPLC erwies sich die Tacrin/MeOH-Probe als die, mit dem interessantesten Aktivitätsprofil. Die Charakterisierung der entstandenen Verbindungen bezüglich ihrer Vielfältigkeit erfolgte mittels ESI-Massenspektrometrie und UV-Spektroskopie. Die Substanz mit höchster Hemmwirkung gegenüber AChE (Peak E der Tacrin/MeOH-Probe) wurde nach der Isolierung der Reinheitsprüfung und Strukturaufklärung mittels NMR-, FTIR- und (Tandem)-ESI-Massenspektrometrie zugeführt und auf ihre biologische Wirkung hin untersucht. / This study aimed at developing and biological screening of new potential acetylcholin-esterase (AChE) inhibitors. They represent the most widely used class of therapeutics for treatment of cognitive disorders such as Alzheimer´s Disease and neuromuscular diseases. The inhibitory potency toward acetylcholinesterase was evaluated by means of the spectrophotometric Ellman test. At first, the series of AChE inhibitors of bispyridinium type derived from TMB4 [1,1´-Trimethylene-bis-(4-formyl-pyridinium bromide) dioxime] was tested against AChE. In essence, the substitution pattern influenced the inhibitory potency against the enzyme. The most potent inhibitor of the bispyridinium-class was the 2,6-halogenated compound DUO3 (IC50 = 0.34 μM), followed by monobenzyl substituted (UNO3), bismethyl substituted (TBM), and unsubstituted derivatives of TMB4. This experimental finding could be explained by docking studies whose goal was to thoroughly explore possible binding modi of this type compounds. One major observation was that almost all of the compounds docked display a general binding mode; they are a kind of ditopic inhibitors which bind to both the catalytic and the peripheral sites of the enzyme. The interactions found after docking include π-π-stacking with amino acid residues of the anionic substrate binding site (Trp84), cation-π contacts with Phe331 and Tyr334 of aromatic gorge and the peripheral anionic binding site (Trp279). This type of interactions is already known for other potent AChE-inhibitor donepezil. Moreover, all compounds were potentially able to bind inside the active side gorge, although, not the whole molecule was able to interact with amino acid residues of the enzyme. This “size problem” of the DUO-ligands may be one reason for their reduced activity as compared to other potent AChE-inhibitors e.g. donepezil, tacrine. Using the hypothesis of the study on bisbenzylethers of bispyridinium compounds which were too long to tightly fit into gorge of AChE, shorter compounds of pyridinium type were developed. This new series of compounds was systematically shortened from 2,6-dichloro-benzyl, 2-chlorobenzyl and phthalimidomethyl ditopic compounds and substituted with several moieties on contralateral end of molecule. The most potent pyridinium compound was 3c (IC50 = 0.073 μM), the activity of which was in the same range of inhibition concentration as tacrine (IC50 = 0.044 μM). In addition, the affinity of 3c toward BChE was rather low (IC50 = 2.49 μM), indicating a lower degree of side effects. In addition to the rational drug design new potent AChE-inhibitors were generated through serendipitous but reproducible approach – via random chemistry. In order to achieve this objective, compound libraries were generated by using gamma-irradiation as an initiator of random free radical recombinations in aqueous or alcohol solution of starting compounds. The bioassay-guided-HPLC-fractionation as a deconvolution strategy of the gained product library was employed. Where the activity in a single first round fraction was observed further steps of fractionation were warranted till a potent hit was observed. The biological screening of first round fractions revealed the tacrine/methanol mixture to be the one with most active fractions toward AChE. Hence, further research efforts were focused on separation, deconvolution, isolation and characterization of biologically active principles. HPLC/ESI-MS (high performance liquid chromatography-electrospray ionisation mass spectrometry), NMR and UV spectroscopy were used to determine the origin and character of generated compounds, first of all that of active ones.

The degradation of phosphate triesters is efficiently catalyzed by organophosphate hydrolases (OPH). While a number of recent studies have focused on optimizing the rate of hydrolysis observed with the native enzyme, no dinuclear complexes that mimic the function of OPH have been reported or investigated. Our present research focuses on the synthesis of dinuclear metal complexes and on the study of their catalytic abilities. An important aspect of this research concerns the investigation of the coordination chemistry of dinuclear ligands designed to hold two metal cations in well defined positions. The ability of the different complexes to catalyze the degradation of thiophosphate triester is presented. Out of several complexes studied, ortho-metallated Pd (II) complexes have been found to display the highest catalytic activity for the hydrolysis of parathion.

The degradation of phosphate triesters is efficiently catalyzed by organophosphate hydrolases (OPH). While a number of recent studies have focused on optimizing the rate of hydrolysis observed with the native enzyme, no dinuclear complexes that mimic the function of OPH have been reported or investigated. Our present research focuses on the synthesis of dinuclear metal complexes and on the study of their catalytic abilities. An important aspect of this research concerns the investigation of the coordination chemistry of dinuclear ligands designed to hold two metal cations in well defined positions. The ability of the different complexes to catalyze the degradation of thiophosphate triester is presented. Out of several complexes studied, ortho-metallated Pd (II) complexes have been found to display the highest catalytic activity for the hydrolysis of parathion.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 132-149). Also available in electronic version. Access restricted to campus users.