Rational design of diflunisal analogues with reduced affinity for human serum albumin

Many lead compounds bind to serum albumin and exhibit markedly reduced effi cacy in vivo as compared to their potency in vitro. To aid in the design of compounds with reduced albumin binding, we performed nuclear magnetic reso nance (NMR) structural and binding studies on the complex between domain II I of human serum albumin (HSA-III) and diflunisal, a cyclooxygenase inhibit or with antiinflammatory activity. The structural studies indicate that the aromatic rings of diflunisal are involved in extensive and specific intera ctions with hydrophobic residues that comprise the binding pocket in subdom ain IIIA. The carboxylic acid of diflunisal forms electrostatic interaction s with the protein similar to those observed in the X-ray structure of HSA complexed to myristic acid. In addition to the structural studies, NMR-deri ved binding constants were obtained for diflunisal and closely related anal ogues to develop a structure-affinity relationship for binding to subdomain IIIA. On the basis of the structural and binding data, compounds were synt hesized that exhibit more than a 100-fold reduction in binding to domain II I of HSA, and nearly a 10-fold reduction in affinity for full length albumi n. Significantly, several of these compounds maintain activity against cycl ooxygenase-2. These results suggest a rational strategy for designing out a lbumin binding in potential drug molecules by using structure-based design in conjunction with NMR-based screening.