Evaluation of micro-parallel liquid chromatography as a method for HTS-coupled actives verification

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• High-throughput screening (HTS) identifies biologically active compounds but requires extensive postscreening analysis.
• Verifying compound activity is crucial to distinguish true hits from artifacts.

Purpose of the Study:

• To evaluate micro-parallel liquid chromatography (microPLC) as a separation-based enzyme assay platform for HTS follow-up.
• To integrate microPLC directly after primary screening for immediate secondary analysis.
• To assess microPLC's performance in verifying compound activities against hydrolase and oxidoreductase targets.

Main Methods:

• Developed a workflow coupling HTS with microPLC for rapid verification of "active" wells.
• Consolidated samples from a 1,536-well HTS plate into a 384-well plate for microPLC analysis.
• Utilized a 24-channel microPLC system for simultaneous high-performance liquid chromatography analysis of up to 24 samples.

Main Results:

• MicroPLC analysis confirmed the activity of two glucocerebrosidase activators.
• Identified several non-biological "positive" samples, including false enzyme activators due to compound fluorescence.
• The method required only 0.6% of the original assay volume (6 microliters).

Conclusions:

• Micro-parallel liquid chromatography is an effective platform for post-HTS secondary analysis.
• MicroPLC significantly reduces the need for extensive postscreening analysis by rapidly identifying false positives.
• The integration of microPLC into screening workflows enhances efficiency and accuracy in compound verification.