AbstractsChemistry

In drug discovery, it is important to use high throughput screening (HTS) technologies to rapidly identify active compounds for biological targets (usually enzymes) from large chemical libraries. The state of art strategy for HTS is coupling multiwell-plates (MWPs) to optical readers. Higher throughput, less reagent use and minimal labeling are always pursued in HTS. Mass spectrometry (MS) is a powerful label-free analyzer due to its high speed and sensitivity. Segmented flow (droplets) can reliably manipulate nanoliter samples and miniaturize reactions with high precision and automation. Novel high throughput screening systems have been developed by interfacing oil-segmented droplets to electrospray ionization (ESI)-MS. To miniaturize a screening, we designed an all-droplet system for conducting assays inside nanoliter droplets. A microfabricated reagent addition device was used for injecting multiple reagents into the droplet array of test compounds to initiate enzymatic reactions. The reaction droplets were directly analyzed by ESI-MS. This all-droplet system was demonstrated by a cathepsin B inhibitor screening with high reliability (Z-factor = 0.8), high analysis rate (0.8 Hz) and straightforward interpretation. Reagents consumption was at picomoles to femtomole level, which is 1000-fold less than the traditional MWP-based assays. Integrating droplet-ESI-MS with existing MWPs screening workflow can extend the application of both systems. With this concept, we developed a ???MS plate reader??? (MSPR). It can reformat 3072 samples from eight 384-well plates into oil-segmented droplets in 13 min (4.5 Hz), and then analyze them in 30 min (up to 2 Hz). Using MSPR, a label-free screen for cathepsin B inhibitors against 1280 chemicals was completed in 45 min (triplicate assay, 1.6 Hz). 11 novel inhibitors were identified and validated. We also developed MS assays for two health beneficial enzymes: SIRT1 and SIRT6. Both assays are applicable to large-scale screenings using MSPR. An 80-compound pilot screening for SIRT1 modulators identified 4 strong inhibitors (> 50% inhibition), all of which were confirmed by dose-dependent experiments. A 25-compound test screening of SIRT6 modulators demonstrated the reliability of this assay by identifying the known activator (> 200% activation). It also showed that the single assay is as robust (Z-factor=0.6) as the replicated assay.