An Ultra-High Throughput Screening Approach for an Adenine Transferase Using Fluorescence Polarization

Abstract

We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated to adenine of the donor substrate through a C6 spacer arm. As a result of the transfer of the adenine-containing moieties to the acceptor protein substrate, the rotational correlation time of the Texas Red conjugate increased, hence increasing the degree of fluorescence polarization. The pharmacological profile and kinetics of the enzyme measured according to the FP method were consistent with those determined previously by conventional analysis. We have successfully executed a 250,000-compound high throughput screening program based on the FP assay method. The quality and validity of the assay were verified by a variety of statistical analyses.

High‐Throughput Screening for Inhibitors of Sialyl‐ and Fucosyltransferases

“…Therefore we reasoned that transfer of a sugar with a fluorescent tag to a suitable glycoprotein acceptor substrate would form the basis of a homogeneous FP assay owing to the large difference in molecular size between the fluorescent substrate and the glycoprotein products (Figure 1). 4…”

High‐Throughput Screening for Inhibitors of Sialyl‐ and Fucosyltransferases

“…Since STs and FUTs are well documented to utilize nucleotide sugar donor substrates modified to contain bulky substituents on the sialic acid at C-9 or fucose C-6, [3] respectively, we reasoned that transfer of a sugar with a fluorescent tag to a suitable glycoprotein acceptor substrate would form the basis of a homogeneous FP assay due to the large difference in molecular size between the fluorescent substrate and glycoprotein products (Figure 1). [4] …”

Inhibiting AMPylation: A Novel Screen To Identify the First Small Molecule Inhibitors of Protein AMPylation

“…In the presence of an inhibitor, however, fluorophore transfer is blocked and there is no increase in FP. 19 We recently identified Fl-ATP as a fluorescently-tagged ATP analogue that is compatible with this type of HTS assay (Figure 1C). Briefly, the k cat / K M of Fl-ATP is only 10-fold lower than ATP (1.0 × 10 4 versus 1.2 × 10 5 M −1 s −1 for Fl-ATP and ATP, respectively), and we verified that this fluorescent nucleotide is transferred to VopS substrates.…”

“…The basis of our assay platform is the enzyme-catalyzed transfer of a fluorophore to a protein substrate, which results in an increase in fluorescence polarization (FP) (Figure ). In the presence of an inhibitor, however, fluorophore transfer is blocked and there is no increase in FP . We recently identified Fl-ATP as a fluorescently tagged ATP analogue that is compatible with this type of HTS assay (Figure C).…”