The unique combination of micro-fluidics and high-throughput mass spectrometry utilized by RapidFire™ Lead Discovery enables us to rapidly develop solution-phase, functional enzyme assays using native, label-free substrates to unlock the value of otherwise intractable targets. It can be used for the direct quantification of analytes in complex reaction mixtures with typical throughput of 4-5 seconds per sample.

Screening results represented as an activity distribution chart. The activity of the assay controls and test compounds (percent inhibition relative to the assay controls) are shown as three separate traces (red trace, negative controls; blue trace, positive controls; green trace, test compounds).

In this case study, RapidFire™ Lead Discovery was used to identify inhibitors of acetylcholinesterase, an enzyme with clinical importance in Alzheimer's disease. Using RapidFire™ high throughput mass spectrometry technology, we screened a small chemical library, identified several potent inhibitors, and determined their IC₅₀ values.

Acetylcholinesterase (AChE) converts acetylcholine to choline, changing the molecular mass from 146.2 amu to 104.1 amu. We developed a conventional HPLC-MS method to purify acetylcholine substrate and choline product from the reaction buffer with a throughput of about 6 minutes per sample. Utilizing this more traditional methodology, we determined linear enzyme kinetics (0.05 - 50 mU/mL) and the substrate K_m (4.9 µM). This HPLC-MS method was then adapted to the RapidFire™ uHTMS (ultra high throughput mass spectrometry) system, using final concentrations of 10 mU/mL enzyme and 2.5 µM substrate, with a throughput of 1.5 seconds per sample. (Note that throughput is assay dependent and is more typically 4 - 5 seconds per sample.)

Using the RapidFire™ HTMS method, we screened four copies of a 960-member library (Gen-Plus from Microsource) in 48 individual 96-well microtiter plates (including eight negative and eight positive controls per plate). All 4608 samples were analyzed in under 2 hours. The average conversion was 18.7% with a standard deviation of 1.6%, corresponding to a coefficient of variability of 8.6%. Of the 48 plates screened, Z' scores of >0.5 were calculated for 46 plates with an average Z' score of >0.7.

The screen identified several lead compounds with potent inhibitory activity, including the previously known AChE inhibitors: edrophonium chloride, tacrine (Cognex), and monohydroxytacrine. The 14 most potent inhibitors were selected for IC₅₀ determinations. Six-point log dilutions starting at 10 µM were prepared in triplicate for each inhibitor. The IC₅₀ plates were analyzed by RapidFire™ mass spectrometry using the same conditions utilized in the primary screen with the same throughput.

Plate by plate Z' scores. The Z' scores were calculated from the eight negative and eight positive controls present on each plate in the screen.