Evaluation of micro-parallel liquid chromatography as a method for HTS-coupled actives verification.
The identification of biologically active compounds from high-throughput screening (HTS) can involve considerable postscreening analysis to verify the nature of the sample activity. In this study we evaluated the performance of micro-parallel liquid chromatography (microPLC) as a separation-based enzyme assay platform for follow-up of compound activities found in quantitative HTS of two different targets, a hydrolase and an oxidoreductase. In an effort to couple secondary analysis to primary screening we explored the application of microPLC immediately after a primary screen. In microPLC, up to 24 samples can be loaded and analyzed simultaneously via high-performance liquid chromatography within a specially designed cartridge. In a proof-of-concept experiment for screen-coupled actives verification, we identified, selected, and consolidated the contents of "active" wells from a 1,536-well format HTS experiment into a 384-well plate and subsequently analyzed these samples by a 24-channel microPLC system. The method utilized 0.6% of the original 6-microl 1,536-well assay for the analysis. The analysis revealed several non-biological-based "positive" samples. The main examples included "false" enzyme activators resulting from an increase in well fluorescence due to fluorescent compound or impurity. The microPLC analysis also provided a verification of the activity of two activators of glucocerebrosidase. We discuss the benefits of microPLC and its limitations from the standpoint of ease of use and integration into a seamless postscreen workflow.
Simeonov
Anton
A
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA. asimeono@mail.nih.gov
Yasgar
Adam
A
Klumpp
Carleen
C
Zheng
Wei
W
Shafqat
Naeem
N
Oppermann
Udo
U
Austin
Christopher P
CP
Inglese
James
J
D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D005189:False Positive Reactions; D005470:Fluorometry; D005962:Glucosylceramidase / Q000032:analysis / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000032:analysis / Q000378:metabolism; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D007202:Indicators and Reagents; D006903:Hydroxymethylglutaryl CoA Reductases; D005962:Glucosylceramidase