Human serum albumin (HSA) acts as a carrier for testosterone, other sex hormones, fatty acids, and drugs. However, the dynamics of testosterone's binding to HSA and the structure of its binding sites remain incompletely understood. Here, we characterized the dynamics of testosterone's binding to HSA and the stoichiometry and structural location of the binding sites using two-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, bis-ANS partitioning, and equilibrium dialysis, complemented by molecular modeling. 2D NMR studies showed that testosterone competitively displaced 18-[ 13C]-oleic acid from at least three known fatty acid binding sites on HSA that also bind many drugs. Binding isotherms of testosterone's binding to HSA generated using fluorescence spectroscopy and equilibrium dialysis were nonlinear and the apparent Kd varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to two independent binding sites; the binding isotherms were most consistent with two or more allosterically coupled binding sites. Molecular dynamics studies revealed that testosterone's binding to fatty acid binding site 3 on HSA was associated with conformational changes at site 6, indicating that residues in in these two distinct binding sites are allosterically coupled. Conclusions There are multiple, allosterically coupled binding sites for testosterone on HSA. Testosterone shares these binding sites on HSA with free fatty acids, which could displace testosterone from HSA under various physiological states or disease conditions, affecting its bioavailability.

Compound ZJ-101, a structurally simplified analog of the marine natural product superstolide A, was previously developed in our laboratory. In the subsequent structure-activity relationship study, a new analog ZJ-109 was designed and synthesized to probe the importance of the lactone moiety of the molecule by replacing the lactone in ZJ-101 with a lactam. The biological evaluation showed that ZJ-109 is about 8–12 times less active against cancer cells in vitro than ZJ-101, suggesting that the lactone moiety of the molecule is important for its anticacner activity.

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