High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE

Characterizing how chemical compounds bind to human serum albumin (HSA) is essential in evaluating drug candidates. Using warfarin as a test system, w e validate the application of BIACORE SPR biosensors to reliably determine binding constants for drug/HSA interactions. The binding responses for warf arin over HSA surfaces were extremely reproducible even though warfarin is small compared to the size of the immobilized protein. At high concentratio ns, warfarin bound at more than one site on HSA, which is consistent with i ts known binding properties. The affinity we determined for the high-affini ty site (K-d(26 degreesC) = 3.7 +/- 1.2 muM), as well as the dissociation r ate constant (k(d)(25 degreesC) = 1.2 s(-1)), are also consistent with bind ing constants determined previously. These results validate the biosensor t echnology and illustrate how BIACORE can be used to study drug/HSA interact ions in a high-resolution mode. Using a set of 10 test compounds, we presen t a protocol for determining equilibrium dissociation constants for HSA in a high-throughput mode. Our method involves working at low compound concent rations and fitting the equilibrium data for all compounds simultaneously. We show that the % bound values determined by SPR correlate with the values determined by solution-based methods. The ability to examine directly the binding of small molecules (130-800 Da), coupled with minimal sample requir ements and automated instrumentation, makes BIACORE technology applicable f or evaluating drug/HSA interactions. (C) 2001 Academic Press.