Fluorescence Polarization

Although the theoretical principles of fluorescence polarization (FP) were described at the beginning of the century [43], the application of FP to drug discovery, and especially to HTS, became popular only recently [10,44-46]. FP measurements permit analysis of molecular orientation and motion of intrinsically fluorescent or fluorophore-labeled molecules in solution. Therefore FP methodol l>

ogy can be applied to different assays measuring direct binding, such as antibody-antigen interaction, compound-protein interaction, DNA-protein interaction, protease activity, and others [3,47-49].

Several phenomena need to be taken into consideration to characterize the basic principle of fluorescent polarization, FP. First, a time interval exists between the excitation of the fluorescent molecule and the emission of light. This fluorescent lifetime (t) is usually in the nanosecond range. Second, molecules absorb and emit light based on the transition moment of the molecular electronic frame-work—where transition moment is defined as the direction in which the molecule most easily absorbs the light; therefore the linear direction of the emitted light is dependent on the direction of the excitation vector. The excitation polarized light and the emitted light will be parallel only if the molecule remains stationary during the fluorescent lifetime. However, if the molecule is freely rotating in solution during its excited state, then the emitted light will be depolarized. The ability to distinguish between polarized and randomly scattered light is the basis of FP. The fluorescence polarization values (P) are determined by the ratio between the difference of perpendicular and parallel emitted light measurements and the sum of those measurements, where parallel direction is defined as a direction of a polarized excitation light vector. The molecules with their excitation dipoles oriented perpendicular to the excitation light vector cannot absorb light and therefore will not emit light when excited. The parallel alignment will result in 100% light absorbtion. Molecules at other orientations will absorb light at different efficiencies depending on their alignment to the excitation light plane. Therefore the above maximum theoretical polarization value is equal to 0.5P (or 500 mP) [3,50,51]. A polarization value greater than 500 mP suggests an experimental artifact, due either to an additional source of scattered light or to incorrect instrument calibration.

Fluorescent probes can be selected based on their lifetime, but molecules with more extensive t values have an advantage because they can rotate more during the excited state, thus causing greater depolarization. Currently, fluores-cein and its derivatives are the fluorophores of choice. However, new fluoro-phores, that possess long lifetimes, have been recently developed, e.g., BODIPY (Molecular Probes, Eugene, OR) or the Cy dyes (Amersham Pharmacia Biotech, Little Chalfont, UK). Because the emission of those fluores is in the red portion of the spectrum, the interference from fluorescent organic compounds is minimal, thus making this fluorophore more suitable for HTS.

The advantage of FP is that the assay is homogenous, eliminating the necessity to separate bound versus unbound fluorescent probes. The reaction usually reaches equilibrium in a few minutes, thus making it even more attractive for assay miniaturization. Moreover, FP is independent of fluorescence intensity and therefore less sensitive to quenching than other fluorescent detection methods. It also is not affected by solution turbidity, because the FP values are obtained as single-wavelength ratiometric measurements. Because FP measures the changes in molecular orientation and motion, there are two major considerations in designing a successful FP assay. First, high binding affinity/avidity between the ''low'' molecular weight fluorescent-labeled ligand and a relatively large receptor, and, second, the interaction between receptor and ligand has to be rigid in order to eliminate the ''propeller effect'' [10].

FP has multiple advantages for use in the miniaturized format. The assay itself is homogenous, without the necessity to separate bound versus unbound fluorescent probes. The measurement is independent of the fluorescent signal intensity, and the limit of FP sensitivity is dependent only on the affinity of the binding between assay components. Accordingly, in FP, as compared to other assay formats, the decrease in assay volume does not translate to a decrease in P values, although the noise may increase. Figure 7 provides an example of a FP assay formatted in a 1536-well plate [52]. In this experiment, the binding between a fluorescein-labeled 8-mer peptide and an 81.1 kD CDK2/E protein complex was measured. The Z' factor of 0.73 indicates that it is an excellent assay to use in uHTS. The %CV across the 1536-well plate of the positive control (maximum signal with protein complex) and negative control (minimum signal without protein complex) are 2.9% and 3.6%, respectively. The specificity of interaction is determined by the competition experiment with nonlabeled tracer

Figure 7 Flourescent polarization measurements of CDK2/E complex and fluorescein-labeled 8-mer peptide (tracer) interaction. Well-to-well variation for positive and negative controls (with and without CDK2/E, respectively) was performed on two separate plates (N = 1536). Solid lines represent the mean ± 3 standard deviations. The coefficient of variation (CV) is 2.9% for the positive control and 11.1% for the negative control. The zZ factor is 0.75.

Figure 7 Flourescent polarization measurements of CDK2/E complex and fluorescein-labeled 8-mer peptide (tracer) interaction. Well-to-well variation for positive and negative controls (with and without CDK2/E, respectively) was performed on two separate plates (N = 1536). Solid lines represent the mean ± 3 standard deviations. The coefficient of variation (CV) is 2.9% for the positive control and 11.1% for the negative control. The zZ factor is 0.75.

1 10 100 1000 10000 Unlabeled Tracer (nM)

Figure 8 Demonstration of fluorescent polarization assay specificity, showing competition binding of unlabeled and fluorescently labeled tracer to CDK2/E. The IC50 is 42 nM, and the hill slope is 1.1.

1 10 100 1000 10000 Unlabeled Tracer (nM)

Figure 8 Demonstration of fluorescent polarization assay specificity, showing competition binding of unlabeled and fluorescently labeled tracer to CDK2/E. The IC50 is 42 nM, and the hill slope is 1.1.

(Fig. 8). These data indicate the suitability of FP for use in the miniaturized format.

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