131P Queen Elizabeth II Conference Centre London
BPS Winter Meeting 2011

 

 

Direct estimation of ligand affinity for HSA drug site 1 and site 2 using SPA binding assays

Cheryl Parry, Penny Ensor, Christoph Zueger, Matthias Frommherz, Carsten Bauer, Steven Charlton, David Sykes. Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham, West Sussex RH12 5AB, Horsham, UK.

 

Human Serum Albumin (HSA) is the most abundant protein in blood plasma, and constitutes 50-60% of total plasma protein. The ability of HSA to bind drug molecules means it can have a significant influence on the concentration of free drug available to bind the target receptor, affecting drug pharmacokinetic-dynamic relationships and dictating drug dosing regimes. Currently, low throughput methods of determining the free drug fraction in serum are generally adopted for predicting clinical dosing. Here we describe a method for determining the affinity (Kd) of compounds for drug site 1 and drug site 2 on HSA using scintillation proximity binding (SPA) assays.

The Kd of [3H]-MRE269, a prostacyclin IP receptor (IPR) agonist and a known drug site 1 binder (Ensor et al., 2010), was determined directly through saturation binding in PBS. Subsequently a fixed concentration of [3H]-MRE269 (3nM), SPA bead (0.2mg/well) and HSA (63.1nM) was used in a 96 well competition binding format to determine the affinity of unlabelled reference ligands for drug site 1. The Kd of [3H]-diazepam which selectively binds drug site 2 was also determined through saturation binding. The affinity of unlabelled compounds for drug site 2 was determined using a fixed concentration of [3H]-diazepam (7.5nM), bead (0.4mg/well) and HSA (3.1μM)…

[3H]-MRE269 saturation binding produced a two site fit, suggesting that MRE269 is bound not only to HSA drug site 1 but at least one other site found on HSA, pKd values of 6.54 ± 0.18 and 5.08 ± 0.13 were estimated. In contrast [3H]-diazepam saturation binding produced a single site fit and a pKd value of 4.41 ± 0.14. The two site fit obtained with [3H]-MRE269 in the saturation analysis was confirmed in the site 1 and 2 competition binding assays (see Table 1). Compound affinity values obtained in these assays were comparable with literature values (Kratochwil et al., 2002) and an indirect filter binding approach utilising IC50 shifts (Ensor et al., 2010). The small % of added [3H]-MRE269 bound to drug site 2 did not affect site 1 compound affinity determinations.

Drug HSA site 1 pKi [3H]-MRE269 HSA site 2 pKi [3H]-diazepam Indirect site 1 pKi Literature pKd
MRE269 6.76 ± 0.10 5.12 ± 0.12 6.21 ± 0.19 nd
RO-1138452 <3.48 3.78 ± 0.08 <4.82 *4.15 ± 0.09
Treprostinil 4.50 ± 0.09 4.39 ± 0.13 nd nd
Warfarin 4.99 ± 0.24 <2.78 5.44 ± 0.11 5.33 ± 0.05
Diazepam <3.48 4.49 ± 0.11 <4 5.31 ± 0.09
Phenylbutazone 6.09 ± 0.08 3.93 ± 0.16 5.67 ± 0.06 5.54 ± 0.11

Table 1. Binding affinity values for prostanoid and reference drugs to HSA. *Ensor et al., 2010, nd = not determined

In conclusion, radioligand binding assays have been developed which can determine the affinity of unlabelled compounds for drug site 1 and drug site 2 of HSA. These assays may have utility in the development of new chemical entities when understanding a compounds serum albumin binding is the key to achieving in vivo efficacy and reducing off-target effects.

 

Ensor P. et al., ( 2010) PA2online 8(1) 150P.

Kratochwil NA. et al., (2002) Biochemical Pharmacology 64, 1355-1374.