Thesis (M.Sc.)--Chulalongkorn University, 2007

Topoisomerase I has been recognized as a biological target for anticancer or antimicrobial agents. The conventional assay of the enzyme for enzyme activity and screening of enzyme inhibitors was typically agarose-gel electrophoresis. The disadvantages of this method were time-consuming and low throughput. Thus, the purpose of this study was to develop assays by using fluorescent dyes and 96-well microplate format to obtain the more simple and rapid method for screening inhibitor. Four fluorescent dyes including Hoechst 33258, Hoechst 33342, Picogreen and Sybr Green I were investigated. From this study, only Picogreen was most effective to separate minimum signal from maximum signal with the Z value above 0.4. Optimal DNA concentration to be used in Picogreen-based assay should be in range of 50-1000 ng/ml to obtain the Z value above 0.4. The time-course study of topoisomerase I activity using Picogreen was performed to compare between gel-based assay and fluorescence microplate assay. The study suggested that the fluorescence microplate assay was more sensitive than gel-based assay. The utility of our assay was comfirmed by mearsuring the effect of known topoisomerase I inhibitors (camptothecin, heparin, quercetin and menadione) and topoisomerase II inhibitors (etoposide and ellipticine). The IC50 values of topoisomerase I inhibitors from fluorescence method were comparable to that from gel-based method, even though the previous assay were higher IC50 value. However, it could not measure the IC50 value of quercetin because of an interference of the fluorescence intensity of Picogreen by quercetin. In this study, ellipticine, an intercalative topoisomerase II inhibitors exhibited inhibitory effect on topoisomerase I was 100% at 20 µM and it also interfere the fluorescence intensity of Picogreen. Tweleve unknown compounds were selected to screen by both assays. From gel-based assay, chelerythrine, sanguinarine, oxostephanine and fagarine could inhibit topoisomerase I at concentration of 50 µM for 41.1±18.9%, 47.7±9.01%, 84.3±23.4% and 106±8.5%, respectively. The limitation of fluorescence microplate assay was inability to detect intercalating agents such as chelerythrine and sanguinarine. In conclusion, we could develop a rapid assay for topoisomerase I inhibitors using Picogreen as fluorescent probes. If the compounds have DNA binding properties, gel-based assay should be used instead of the fluorescence microplate assay.