Protein Binding

Study design

In vitro protein binding studies are used to estimate the fraction of an asset that remains unbound to plasma and brain proteins. Binding to rat plasma and rat brain homogenate proteins is assessed using in vitro rapid equilibrium dialysis (RED) and/or Centrifree ultrafiltration (Bhatt et al. 2020, Reddy et al. 2022). In both assays, the asset is incubated with a protein matrix (rat plasma or rat brain homogenate), the proteins bound and unbound to the asset are separated into individual fractions, and the amount of asset in each fraction is measured. Additionally, a sample in which the asset is incubated with the protein matrix but the fractions are not separated is used as a control to determine the recovery of the asset in the protein matrix after incubation. The RED assay is used first and Centrifree may be added to better understand the potential protein binding of the asset, particularly in cases when recovery of the asset is low in the RED assay. In both assays, standards, including acebutolol, quinidine, and/or warfarin, may be tested alongside the asset as a quality control measure to ensure their protein binding values match the values found in previous assessments. For the assessments of PSPP controls, values were compared with published literature, and standards were included intermittently to ensure reproducibility of the methodology.

Rapid Equilibrium Dialysis (RED)

A protein matrix (rat plasma or rat brain homogenate) is spiked with the asset (in DMSO, with a final concentration of 1% DMSO) then loaded into a dialysis chamber opposite a phosphate-buffered saline buffer, separated by a 8 kDa membrane, and incubated for 4 hours at 37°C. Samples are taken from each chamber, prepared for analysis, then assessed with high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) using parameters specific to the asset being detected. The amount of asset remaining in the protein chamber and the buffer chamber are used to calculate the amount of asset bound and unbound to the protein matrix.

Centrifree Ultrafiltration

A protein matrix is spiked with the asset then added on top of a filter (30 kDa), incubated for 20 minutes at 37°C, and centrifuged for 10 minutes. Samples are taken from the top and bottom of the filtration device, prepared for analysis, then assessed with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using parameters specific to the asset being detected. The amount of asset in the compartments on each side of the filter are used to calculate the amount of asset bound and unbound to the protein matrix.

This work was conducted by PsychoGenics Inc. (Paramus, NJ) in collaboration with PSPP, NINDS, NIH under contract # 75N95019D00026