Supramolecular displacement-mediated activation of a silent fluorescence probe for label-free ligand screening

Abstract

We report a new approach for the rapid screening of analyte binding affinities for a target protein. We demonstrate that a molecular probe, with a pro-fluorophore substrate and ligand moieties, can be hindered from enzymatic access when bound to the target protein. When analytes displace the probe from the protein's binding pocket, a fluorescence profile is generated. This profile is used to discriminate analytes based on their relative binding affinities.

Figure 1

a) Schematic illustration of the strategy for label-free ligand screening and b) probe structure.

Figure 2

Hindering enzymatic action upon the probe molecule, due to protein binding. a) hCA1 unable to hinder hydrolysis on the probe with a linker n=8 and, b) hCA1 hinders hydrolysis on the probe with a linker n=4. Probe concentrations in all measurements was 5 μM.

Figure 3

Displacement-mediated activity profiles generated by (a) the analytes 1, 2, 4–7; and (b) 1, 8, 3, 4 with the slopes of the curve corresponding to the effective probe displacement by the analytes (experimental parameters on page S4).

Figure 4

Correlation between literature-reported binding constants and slopes. Data from Figure 3a (see Table S1).

Figure 5

Probe (n=4) displacement-mediated activity profiles generated by the analytes 1, 3, 4, 6, 7 with 96-well plate reader.

Scheme 1

Synthesis of the probe

Chart 1

Structure of the analyte molecules used in the assay and their literature-reported binding affinities.