Selective inhibition of acetylcholinesterase 1 from disease-transmitting mosquitoes Design and development of new insecticides for vector control

iv Swedish summary v List of publications vi List of abbreviations viii 1. Introduction 1 1.1. Infectious diseases 1 1.2. Vector-borne infectious diseases 2 1.2.1. Malaria 2 1.2.2. Dengue 3 1.2.3. Other severe mosquito-borne diseases 4 1.3. Vectors 4 1.3.1. Mosquitoes as disease-vectors 5 1.3.2. Anopheles mosquitoes 5 1.3.3. Aedes mosquitoes 6 1.4. Vector control 7 1.4.1. Insecticide independent vector control strategies 7 1.4.2. Insecticide-based vector control interventions 7 1.4.3. The limited number of recommended insecticides 8 1.4.4. Insecticide resistance – a true setback 8 1.4.5. Additional concerns with current insecticides 9 1.5. Acetylcholinesterase 9 1.5.1. The structure of AChE 10 1.5.2. AChE1 of mosquitoes 11 1.5.3. Insecticide insensitive AChE1 12 1.5.4. Current development of AChE1 inhibitors 12 2. Objectives 13 3. Methods and assays 14 3.1. Production of AChE1 14 3.1.1. AChE1 constructs 14 3.1.2. AChE1 expression 14 3.1.3. AChE1 purification 15 3.2. Assays to monitor AChE activity 15 3.2.1. Ellman assay 15 3.2.2. Choline oxidase assay 16 3.2.3. Enzyme kinetics 17 3.2.4. Inhibition studies 18 3.3. Small organic molecules as chemical tools 18 3.3.1. Hit discovery by high throughput screening 18 3.3.2. Design and use of synthesized analogues 19 3.3.3. Evaluation of unwanted toxicity of compounds 19 3.4. Methods to study AChE structure 20 3.4.1. Protein crystallization 20 3.4.2. X-ray data collection and structure refinement 20 3.5. In vivo studies 21 3.5.1. Mosquito rearing 22 3.5.2. Insecticidal effect of compounds 22