An automated high throughput filtration assay: application to polymerase inhibitor identification.
A high throughput filtration assay was developed and automated for screening compounds and natural product extracts using 96-well filterplates. The selection of a hydrophobic membrane and appropriate capture conditions enabled us to perform the enzymatic reaction, capture of products, vacuum filtration, and detection in a single filtration microplate, without any transfer step. The hydrophobic membrane has the advantage of preventing any loss of aqueous solution during the enzymatic reaction. The results are comparable with those obtained in a solid plate format followed by transferring the reaction mixture to a traditional hydrophilic filterplate. This assay is being used to screen for microbial RNA polymerase inhibitors as potential new drugs for treatment of emerging antibiotic-resistant bacterial and fungal infections. The filtration setup can be applied to a variety of assay systems.
Wu
P
P
Tularik Inc., South San Francisco, California 94080, USA. wu@tularik.com
Daniel-Issakani
S
S
LaMarco
K
K
Strulovici
B
B
D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005374:Filtration / Q000295:instrumentation* / Q000379:methods*; D008567:Membranes, Artificial; D012313:RNA / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012371:Robotics; D014158:Transcription, Genetic
D004791:Enzyme Inhibitors; D008567:Membranes, Artificial; D012313:RNA; D003570:Cytidine Triphosphate; D004247:DNA; D012321:DNA-Directed RNA Polymerases