Estimation of plasma protein binding affinities of IP receptor ligands using a radioligand receptor binding assay

Penny Ensor1, David Sykes1, Daniel Blakeley1, Carsten Bauer2, Bernard Kohler2, Peter Leupp2, Christoph Zueger2, Catherine Leblanc1, Steven Charlton1. 1Novartis Institutes for Biomedical Research, Horsham, United Kingdom, 2Novartis Institutes for Biomedical Research, Basel, Switzerland.

Human serum albumin (HSA) is the most abundant extracellular protein found in blood plasma (Mr 66 kDa, 0.53-0.75mM). It binds a variety of structurally diverse drug molecules, exerting a profound effect on the availability of free drug that can bind the target. Traditionally serum protein binding is assessed by determining free fraction in serum using low through-put methods such as equilibrium dialysis and ultrafiltration. Here we describe a method for estimating the equilibrium dissociation constant for serum albumin (eKp) using a conventional receptor binding assay.

Firstly, a mathematical model was developed to investigate the influence of plasma protein on ligand-receptor binding. Simulations using this model predicted that Schild analysis was sufficient to determine Kp values provided the protein concentration remained in excess of the ligand concentration. We chose the human prostacyclin IP receptor (IPR) transfected into CHO cells as a model system to investigate this experimentally, using the radiolabelled antagonist [3H]-RO-1138452 (Clark et al, 2004). Schild plots were constructed for saturation binding of [3H]-RO-1138452 (0.01 - 20 nM) in the presence and absence of serum albumin. Increasing concentrations of protein gave parallel shifts in [3H]-RO-1138452 binding with Schild slopes equal to unity. This behaviour was observed for serum albumin from rat, bovine and human, although the estimated affinity was different for each species. We then tested the utility of plasma protein shift assays to estimate the affinity values for the unlabelled IPR agonists iloprost and MRE-269, using the following relationship: epKp = log(DR-1)-log[albumin] at a fixed concentration of [3H]-RO-1138452 (0.6nM) and serum albumin (0.1%). As was observed for [3H]-RO-1138452, the estimated affinities of MRE269 were different depending upon the species of albumin tested (Table 1).

Table 1. Binding affinity values for prostanoid ligands to different species of serum albumin.

In conclusion we have demonstrated the utility of radioligand binding assays for determination of pKp values. In addition we have identified differences in the apparent pKp of IPR ligands for different species of serum protein. These findings could have important consequences for the in vitro and in vivo testing of novel IPR ligands.

Clark et al., (2004) Bioorganic & Medicinal Chemistry Letters 14, 1053-1056.