April 15, 2006 (Vol. 26, No. 8)

Real-time Kinetic Assays for Kinases, Phosphatases, and Proteases

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&#37 pure as determined by HPLC.

The substrate Km for each target was measured by assembling 60-&#181L 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&#176C and 50&#37 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.

Fig.1: Data traces from a four-sipper chip showing varying amounts of substrate conversion to product.

Fig.2: Measurement of K(m) for ATP for AurorA kinase

Linearity of the Reaction

Most HTS enzymatic screening campaigns are run using endpoint assays. Typically the incubation times selected are between 30 minutes and two hours to accommodate automation. Accurate determination of compound potency requires that the endpoint chosen lie within the linear portion of the enzyme&#180s kinetic curve.

Using optimized conditions 60-&#181L reactions containing 1.5-&#181M peptide substrate and the appropriate concentration of enzyme were assembled in wells A1, A5, E1, and E5 of a 384-well microtiter plate. The plate was immediately placed on the LC3000 and samples were introduced onto a 4-sipper chip every five minutes. Temperature and humidity in the reaction chamber were maintained at 20&#176C and 50&#37 respectively.

Peptide and product were separated and detected using the LC3000. The data represents the average std error of the product formed of the four replicate wells over time. Figure 3 shows the time course for each enzyme.

Fig.3: Time course of enzyme activity for all four enzymes in this study

Inhibitor IC(50)s

Determining IC50 values for known inhibitors of each enzyme completed assay development. 31-&#181L reactions containing 1.5-&#181M peptide substrate and the appropriate concentration of enzyme were incubated in the presence of decreasing concentrations of inhibitors. The reactions were stopped by the addition of: AurA kinase-20mM EDTA; MMP9 protease-20mM EDTA and 0.5 mM MMP9 Inhibitor 1; PTP1b phosphatase-2 mM sodium orthovanadate.

Peptide and cleaved product were separated and detected using the LC3000. IC50 values were calculated using nonlinear regression sigmoidal dose-response-variable slope.

The IC50 plots for the protease MMP9 are shown in Figure 4. The IC50s obtained for the kinase AurA and phosphatase PTP1b are consistent with published values.

Microfluidic assays have been developed for three important drug target classes using the Mobility-Shift Assay kinetic format on the LC3000 platform. Assay development and optimization was simplified and accelerated by the ability to follow the reactions in real-time. Actual Km and Ki measurements were also derived by the kinetic measurement of initial rates of product formation, and reaction linearity was directly monitored.

In addition to the benefits of acquiring enzymatic data in a kinetic fashion, the direct and precise measurement of both product and substrate concentration for each reaction improves data quality by providing ratiometric data analysis, resulting in lower false positive and negative artifacts and the ability to detect even low-potency inhibitors, missed by other assay technologies.

Fig.4: IC(50) curves of five known protease inhibitors against MMP9

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