Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells

Abstract

The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (K d ) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular K d values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular K i values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular K i values matched known biochemical K i values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.

Comparative Analysis of the Techniques for the Determination of Binding Affinity between a Small Molecule Inhibitor and a Protein Target

“…Consequently, as an important parameter, binding affinity constant (𝐾 ) has been a valuable in vitro measure for various preclinical evaluation, including the selection of lead compounds, the optimization of leads, and the predictions of in vivo potency, pharmacokinetic properties and safely profiles [5]. Therefore, it is of great important to obtain accurate 𝐾 values of the inhibitors.…”

Synthesis of Monofluorinated 7-Hydroxycoumarin-3-Carboxamides as Cell-Permeable Fluorescent Molecular Probes

“…As an alternative method for studies of target engagement, we recently reported the fluorescent probe cellular binding assay (FPCBA). This new method uses flow cytometry for quantitative studies of the binding of cell-permeable fluorescent probes and competitors to specific native (untagged) proteins that are overexpressed in living cells.…”

“…This new method uses flow cytometry for quantitative studies of the binding of cell-permeable fluorescent probes and competitors to specific native (untagged) proteins that are overexpressed in living cells. Although FPCBA was previously validated using allosteric activators of Protein Kinase C isozymes, our previous studies − of interactions of fluorescent derivatives of the anticancer drug paclitaxel (Taxol) with microtubules in living cells were instrumental in developing this method. Key to this approach is the use of cell-permeable fluorophores that can be efficiently detected by flow cytometry when linked to ligands of protein targets.…”

“…These esters were treated with 1-aminohexane to afford N -hexyl amides ( 26 – 28 ) for subsequent analysis as standards by optical spectroscopy. We previously reported the analogous N -hexyl amides of 7OHCCA ( 29 ) and PB ( 30 ) for comparison.…”

“…Absorbance, emission, and quantum yields were measured in PBS (1% DMSO). Molar extinction coefficients for 29 and 30 were previously described . The QY shown for 30 is based on a previously reported PB-biotin amide.…”