Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening

INTRODUCTION Variety of scientific advances and economicpressures in recent years called for improved drugdiscovery screening technology (Burbaum, 1998;Major, 1998). In response to that call, a number ofpotential therapeutic targets from the field offunctional genomics have emerged and largecompound libraries derived from parallel andcombinatorial chemical synthesis techniques havebeen developed. These new advances have helpedthe pharmaceutical industry reduce developmentcosts and e…

Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening

A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concen- tration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detec- tion (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.