High-throughput Screening Studies of Inhibition of Human Carbonic Anhydrase Ii and Bacterial Flagella Antimicrobial Activity
Year: 2010
Type: dissertation
Abstract: TABLES 2.1.Potential carbonic anhydrase II inhibitor compounds identified in initial screen of Genesis Plus compound library, as confirmed by two statistical criteria 49 2.2.Summary of IC50 values and K& values for Genesis Plus hit compounds and model sulfonamide compounds 74 2.3 Carbonic anhydrase II inhibitor compounds identified in a screen of the NCI Diversity set compound library 83 3.1.Structures and apparent IC50 values for inhibition of human carbonic anhydrase II by thioxolone analogs 119 3.2.Michaelis-Menten kinetic parameters determined for concentrationdependence of thioxolone inhibition of 4-nitrophenyl acetate hydrolysis by carbonic anhydrase II 131 3.3.Mechanism-based enzyme inactivation parameters determined for thioxolone inhibition of 4-nitrophenyl acetate hydrolysis by carbonic anhydrase II 136 4.1.Complete list of antibacterial compounds detected in screen of Genesis Plus library with offset motility detection method 161 5.1.Mammalian a-class carbonic anhydrase clone vector and sequence information 170 5.2.Sequencing results for the pET28c-CA I and pET28c-CA IV 176 5.3.Partial blastx search results with CA I DNA sequence results 177 5.4.Partial blastx search results with CA IV DNA sequence results 178 5.5.The translated peptide sequence for the subcloned carbonic anhydrase I and IV genes in the pET 28c-CA I and pET 28c-CA IV plasmids 179 vii List of Tables-Continued 5.6.Comparison of the translated CA I pET28c-CA I plasmid DNA sequence to the wild-type protein sequence 180 5.7.Comparison of pEt 28c-CA I V plasmid DNA sequence to the wild-type CA IV sequence 181 5.8.Results of non-linear fit of standard CA II esterase assay IC50 data using Graph Pad Prism 4.0 sigmoidal dose-response model/curve fit 206 5.9.Results of non-linear fit for microplate format CA II CO2 hydration pH assay IC50 data using Graph Pad Prism 4.0 sigmoidal dose-response model/curve fit 206 5.10.Statistical results of fluorescein MultiDrop 8-channel peristaltic test for three successive dispensing operations into 96-well microplates 209 5.11.The concentration of reagents right after mixing the aqueous solution of sulphuric acid, cobalt sulphate, and potassium dihydrogen phosphate with an aqueous solution of sodium bicarbonate 210 5.12.CA II carbonic acid dehydrating to CO2 pH assay.Assay components and concentrations 212 5.13.Comparison of standard 4-nitrophenyl acetate CA II esterase assay vs. CA II carbonic acid dehydrating to CO2 pH assay 213 viii LIST OF FIGURES Ribbon diagram of human alpha-class carbonic anhydrase II enzyme structure 5 The active site of HCA II with the active site residues as labeled and the zinc atom shown as a grey sphere 6 An example of p-carbonic anhydrase from the dicotyledonous plant Pisum sativum 9 y-carbonic anhydrase from Methanosarcina thermophila, with the zinc ions as grey spheres and the three histidine residues that coordinate the zinc (His 81, His 117, and His 122) shown in stick form 12 The first ^-class of carbonic anhydrase (CDCA1), from the marine diatom Thalassiosira weissjlogii 14 Chemical structure of acetazolamide, a sulfonamide inhibitor of carbonic anhydrase II, molecular formula C4H6N4O3S2 15 Complete structure of flagellin protein from Salmonella typhimurium 20 Bacterial flagella fibers and flagellin protein 21 Plot of example 4-NPA esterase IC50 data fit to Eq. 2.1 for two sulfonamide and two non-sulfonamide compounds identified as carbonic anhydrase inhibitors in the Genesis Plus compound library 44 Initial results of screening the Genesis Plus 960 compound library for inhibitors of human CAII 48 Analysis of assay quality for each of the 12 compound plates in the Genesis Plus collection that were screened for inhibition of carbonic anhydrase 4-NPA esterase activity 56 Example of raw kinetic absorbance data simultaneously collected for eight compounds (column 8, plate 1) during initial screen of compound library for CA II inhibitors 58 ix XI List of Figures-Continued 5.3.Plasmid map of pET28c-CA IV, showing the CA IV gene, the kanamycin resistance gene, and the lac I gene 183 5.4.SDS-PAGE analysis of CA I and CA IV protein expression using the BL21-AI strain of E. coli 186 5.5.Plot of the initial FPLC purification run of CA IV protein using the AKTA™ FPLC instrument with the FPLC UNICORN™ software 190 5.6.Plot of the FPLC purification run of CA I protein using the AKTA™ FPLC instrument with the FPLC UNICORN™ software and a 50 ml SP Sepharose FF column 191 5.7.Plot of the first step of the FPLC purification of Cam protein using the AKTA™ FPLC instrument with the FPLC UNICORN™ software 195 5.8.Plot of the second step of the FPLC purification of Cam protein via hydrophobic interaction chromatography 196 5.9.Bicarbonate dehydration kinetic absorbance assay results for CA II -10 mM NaHC0 3 pH assay 198 5.10.Bicarbonate dehydration kinetic assay results for CA II -60 mM NaHC03 pH assay 200 5.11.Bicarbonate dehydration kinetic assay results for Cam -60 mM NaHC03 pH assay with serial dilution of Cam inhibitors 202 5.12.Comparison of CA II microplate format CO2 hydration pH assay IC50 vs. standard IC50 esterase assay with 1:2 diluted acetazolamide 205 5.13.Average fluorescein fluorescence emission value for each column 209 5.14.IC50 plots for A. acetazolamide, B. sulfanilamide, and C. ethoxyzolamide using the CA II carbonic acid dehydrating to CO2 pH assay 214 Xll
Authors: Albert A. Barrese Iii, Ph. D
Institution: Western Michigan University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3150909464