With the recent advances in genomics, combinatorial chemistry, and automation, drug discovery scientists working in pharmaceutical, biotechnology, and academic environments are faced with the overwhelming task of developing many new assays. These assays must be robust, rapid, economical, miniaturized, automated, sensitive, and precise.
Traditional assay formats, particularly those used for enzymatic screening, can be quite complex and many require specialized reagents, such as radiolabeled substrates, specific antibodies, or other detection components. In addition, the characterization of enzyme activity through accurate determination of substrate Km and inhibitor Ki requires the ability to measure the rates of product formation. Many of the methodologies used for identifying active compounds that modulate enzymatic activity are limited to endpoint assays and cannot easily be used to follow a reaction over time.
The Caliper Life Sciences (www.caliperls.com) Mobility-Shift Assay format combines the basic principles of capillary electrophoresis in a microfluidic environment to analyze enzymatic assays without the addition of a stop or quenching reagent. The Caliper LabChip (LC) 3000 platform employs microfluidic chips, containing a network of miniaturized, microfabricated channels through which fluids and chemicals are moved to perform experiments.
Two different methods, electrokinetics and pressure, are exerted on the chip to generate fluid motion through the microchannels on the chip. Using vacuum pressure, reactions occurring in 96- or 384-well microplates are introduced, or sipped, through the fused silica sippers (4 or 12 sippers per chip), located in the bottom of the chip. By applying an electric-potential difference across the separation channel, fluorescently labeled substrates and products are separated, based on mass or charge differences, by electrophoresis and detected by laser-induced fluorescence.
Both the substrate and the formed product are detected and measured for each sample. The amount of product formed is determined by calculating the ratio of the product peak/(product + substrate peaks). The data signature of a typical Mobility-Shift Assay is shown in Figure 1. For kinetic analysis the reaction is monitored as it progresses by sequentially sipping samples onto the chip at various time intervals.
A large percentage of the effort to find new drugs has focused on kinases, phosphatases, and proteases. Many of these targets have been demonstrated to play important roles in the modulation of cell metabolism and disease. To date, a large number of Mobility-Shift Assays for these three target classes have been developed on the LC3000 microfluidic platform using real-time kinetics.
We have chosen Aurora A (AurA), Protein Tyrosine Phosphatase 1B (PTP1b), and Matrix Mellatoproteinase 9 (MMP9) as representative enzymes for each target class. Initial velocities were used to calculate substrate Kms for each assay.
The peptides were synthesized with a fluorescein molecule at the amino-terminus position (Tufts University Core Facility) and were greater than 95% pure as determined by HPLC.
The substrate Km for each target was measured by assembling 60-µL reactions, containing increasing amounts of peptide substrate and the appropriate concentration of enzyme in a 384-well microtiter plate. Once assembled, the microplate was immediately placed in the LC3000 instrument. The reactions were then sampled into the chip every five minutes for two hours. Temperature and humidity in the reaction chamber were maintained at 20°C and 50% respectively. Substrate and product were separated and detected using the LC3000.
Initial rates, Vi (ng/min) were determined from the slopes of the linear regression plots at each peptide concentration of product formed in the reaction over time. Km was calculated from nonlinear regression analysis using the Michaelis-Menton equation. The ATP Km plots for the kinase AurA is shown in Figure 2.