Pyrethroids are the only class of insecticides approved by the World Health Organization (WHO) for use in insecticide treated nets (ITNs), the first line of malaria vector control. Widespread resistance development to pyrethroids undermines current control efforts, and hence an urgent need for alternative chemistries. I report the evaluation of pharmacological differences between insect and vertebrate acetylcholinesterase (AChE) as well as selectivity and toxicity testing of new carbamate insecticides on Anopheles gambiae, the African malaria mosquito. AChE gorge pharmacology data revealed differences between insect and vertebrate AChE that can be exploited in the design of a bivalent insecticide. Toxicokinetic analysis showed that metabolic detoxication and cuticular penetration affect toxicity of carbamates in a manner dependent on the chemical structure. Structure activity relationships of side-chain branched N-methylcarbamates emphasized the importance of structural complementarity of ligands to the AChE catalytic active site and the substrate, acetylcholine. Monovalent pyrazoles and acetophenone oxime carbamates were toxic to both susceptible and carbamate-resistant mosquitoes carrying a G119S mutation within the catalytic site. A bivalent phthalimide-pyrazole carbamate and sulfenylated phenyl N-methyl carbamates were highly toxic when topically applied onto insect but less toxic by treated filter paper assays. In vitro evaluation of a molecular mosquito-selectivity model using AChE peripheral site ligands confirmed that selectivity of PRC 472 was due to presence of I70 in mosquito, which is Y70 in human AChE. The findings presented here are important steps in the on-going search of a mosquito-selective and resistance mitigating carbamate insecticide for control of malaria mosquitoes. / Ph. D.

This thesis deals with effects of some chosen 7-methoxitacrine (7-MEOTA) analogues on enzymatic activity of acetylcholinesterase (AChE). 7-MEOTA is a derivative of tacrine, which had been used for symptomatic treatment of Alzheimer's disease (AD), until drugs with better therapeutic index were developed. 7-MEOTA the same way as tacrine therapeutically acts by inhibition of acetylcholinesterase and a neurotransmitter acetylcholine rise in the organism. It shows similar strength and type of inhibition, but it's less toxic contrary to tacrine. Some of the previously examined analogues of 7-MEOTA were as strong or even stronger AChE inhibitors than 7-MEOTA and so promising future medicaments. However, all the compounds analyzed in this thesis showed weaker enzymatic reaction inhibition and AChE affinity. For each of the examined compounds IC50, Ki and Ki' were calculated and AChE inhibition type was determined. All the 7-MEOTA analogues showed a mixed type of the inhibition. The theoretical part of this thesis deals with manifestations and origins of AD, its genetic factors etc. and tries to show some of the anthropological findings a theories connected with the theme.

Os receptores nicotínicos de acetilcolina (colinérgicos) (nAChRs) neuronais são proteínas integrais de membrana e pertencem à família de canais iônicos controlados por ligante, compostos por subunidades α e β. Esses receptores desempenham um papel-chave na transmissão de sinal entre os neurônios nos sistemas nervoso central e periférico. O subtipo α3β4, por exemplo, é o nAChR neuronal mais expresso no sistema nervoso autônomo; nAChRs contendo a subunidade α3 estão presentes em alta densidade no gânglio cervical superior, glândulas pineal e adrenais. Também estão presentes na substancia nigra, striatum, hipocampo, locus ceruleus, tracto habênulo-interpeduncular e cerebelo. Os nAChRs são inibidos por uma variedade de substâncias químicas, incluindo toxinas naturais, anestésicos locais, drogas de abuso (por, exemplo, cocaína) e compostos clinicamente importantes (tranqüilizantes, por exemplo). O mecanismo de inibição desses receptores tem sido investigado intensivamente. Neste estudo, nós investigamos o mecanismo pelo qual a tacrina (9-1,2,3,4-tetraidroaminoacridina), um agente usado clinicamente no tratamento da doença de Alzheimer, inibe o nAChR α3β4 de rato recombinante expresso nas células KXα3β4R2, utilizando uma técnica de cinética química rápida. A constante de dissociação da nicotina do sítio que controla a ativação do receptor, Kd, é 23 µM e a constante de equilíbrio de abertura do canal, Φ-1, é 4. A tacrina inibe o receptor competitivamente, com um KI de 0,77 µM. / Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are integral membrane proteins and belong to the family of ligand-gated cation channels composed by α and β subunits. These receptors play a key role in the signal transmission between neurons in the central and peripheral nervous system. The α3β4 subtype, for example, is the most expressed neuronal nAChR in autonomic ganglia; α3-containing nAChRs are present at particularly high density in the superior cervical ganglia, pineal, and adrenal glands. They are also present in the substancia nigra, striatum, hippocampus, locus ceruleus, habenulo-interpeduncular tract and cerebellum. The nAChRs are inhibited by a variety of chemical substances, including natural toxins, local anesthetics, abused drugs (e.g., cocaine) and clinically important compounds (e.g., tranquilizers). The mechanism of inhibition of these receptors has been intensively investigated. In this study, we investigated the mechanism by which tacrine (9-1,2,3,4-tetahydroaminoacridine), an agent used clinically to treat Alzheimers disease, inhibits the recombinant rat α3β4 nAChR expressed in KXα3β4R2 cells, using a rapid chemical kinetic technique. The nicotine dissociation constant for the site controlling receptor activation, Kd, is 23 µM and the channel-opening equilibrium constant, Φ-1, is 4. Tacrine inhibits the receptor competitively, with a KI of 0.77 µM.

Os receptores nicotínicos de acetilcolina (colinérgicos) (nAChRs) neuronais são proteínas integrais de membrana e pertencem à família de canais iônicos controlados por ligante, compostos por subunidades α e β. Esses receptores desempenham um papel-chave na transmissão de sinal entre os neurônios nos sistemas nervoso central e periférico. O subtipo α3β4, por exemplo, é o nAChR neuronal mais expresso no sistema nervoso autônomo; nAChRs contendo a subunidade α3 estão presentes em alta densidade no gânglio cervical superior, glândulas pineal e adrenais. Também estão presentes na substancia nigra, striatum, hipocampo, locus ceruleus, tracto habênulo-interpeduncular e cerebelo. Os nAChRs são inibidos por uma variedade de substâncias químicas, incluindo toxinas naturais, anestésicos locais, drogas de abuso (por, exemplo, cocaína) e compostos clinicamente importantes (tranqüilizantes, por exemplo). O mecanismo de inibição desses receptores tem sido investigado intensivamente. Neste estudo, nós investigamos o mecanismo pelo qual a tacrina (9-1,2,3,4-tetraidroaminoacridina), um agente usado clinicamente no tratamento da doença de Alzheimer, inibe o nAChR α3β4 de rato recombinante expresso nas células KXα3β4R2, utilizando uma técnica de cinética química rápida. A constante de dissociação da nicotina do sítio que controla a ativação do receptor, Kd, é 23 µM e a constante de equilíbrio de abertura do canal, Φ-1, é 4. A tacrina inibe o receptor competitivamente, com um KI de 0,77 µM. / Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are integral membrane proteins and belong to the family of ligand-gated cation channels composed by α and β subunits. These receptors play a key role in the signal transmission between neurons in the central and peripheral nervous system. The α3β4 subtype, for example, is the most expressed neuronal nAChR in autonomic ganglia; α3-containing nAChRs are present at particularly high density in the superior cervical ganglia, pineal, and adrenal glands. They are also present in the substancia nigra, striatum, hippocampus, locus ceruleus, habenulo-interpeduncular tract and cerebellum. The nAChRs are inhibited by a variety of chemical substances, including natural toxins, local anesthetics, abused drugs (e.g., cocaine) and clinically important compounds (e.g., tranquilizers). The mechanism of inhibition of these receptors has been intensively investigated. In this study, we investigated the mechanism by which tacrine (9-1,2,3,4-tetahydroaminoacridine), an agent used clinically to treat Alzheimers disease, inhibits the recombinant rat α3β4 nAChR expressed in KXα3β4R2 cells, using a rapid chemical kinetic technique. The nicotine dissociation constant for the site controlling receptor activation, Kd, is 23 µM and the channel-opening equilibrium constant, Φ-1, is 4. Tacrine inhibits the receptor competitively, with a KI of 0.77 µM.

In preclinical research on Alzheimer's disease pharmacotherapy, attention is paid to multipotent compounds, enabling intensification of the effect by targeting multiple pathophysiological mechanisms. The aim of the thesis was to assess the effect of multipotent compounds and combination therapy in models of cognitive deficit in the rat. The mechanism of action of the tested compounds was modulation of neurotransmitter systems. The aim of the first part of the study was to compare the effect of experimental monotherapy and combination therapy with an N-methyl-D-aspartate (NMDA) receptor antagonist and a γ-aminobutyric acid type A (GABAA) receptor positive modulator in the trimethyltin-induced model. Superiority of the combination therapy was proven by histological analysis of hippocampal neurodegeneration; however, it did not reach statistical significance in the cognitive test. The other part of the thesis focused on multipotent tacrine derivatives. We demonstrated a positive effect of 6- chlorotacrine-6-nitrobenzothiazole hybrid, as well as 6-chlorotacrine-L-tryptophan hybrid, acting as acetylcholinesterase inhibitors, in the scopolamine-induced model of cognitive deficit. Besides, we demonstrated a low risk of serious side effects of other tacrine derivatives acting as NMDA receptor antagonists....