Binding Analysis of 1α- and 17α-Dihydrotestosterone Derivatives to Homodimeric Sex Hormone-Binding Globulin

Binding studies of the interaction of immobilized 1α- and 17α-aminoalkyl derivatives of 5α-dihydrotestosterone (DHT) with purified N-deglycosylated homodimeric human sex hormone-binding globulin (SHBG) were performed using a surface plasmon resonance biosensor. These 1α- and 17α-derivatives with spacers of appropriate lengths between the amine function and the steroid ring skeleton enabled privileged, sterically undisturbed, interactions of either the 17- or 3-characteristic functional groups of DHT with SHBG. The association constants (K_a1) for the binding of these immobilized DHT derivatives to the first binding site of SHBG, determined by SPR measurements, were 0.16 × 10⁷ M^-1 for 17α-aminopropyl-17β-hydroxy-5α-androstan-3-one (1), 1.64 × 10⁷ M^-1 for 17α-aminocaproyl-17β -hydroxy-5α-androstan-3-one (2), and 1.2 × 10⁸ M ^-1 for 1α-aminohexyl-17β-hydroxy-5α-androstan-3-one (3). These values were compared with global K_a data for the corresponding nonimmobilized DHT derivatives from equilibrium measurements using competitions with a tritiated testosterone tracer: the K_a values were 1.25 × 10⁷ M^-1 for 1, 1.50 × 10⁷ M^-1 for 2, and 140 × 10⁷ M ^-1 for 3, confirming a remarkably high binding affinity of this latter compound for SHBG. A global fitting analysis of the biosensor data revealed that the interaction of the three immobilized steroids with SHBG was best described by a kinetic model assuming two structurally independent binding sites. This hypothesis of a bivalent binding model was also directly suggested by a dual fluorescent signal observed by the flow cytometry analysis of SHBG immobilized as a hybrid complex binding simultaneously two 1α-aminohexyl DHT ligands, one formed by 3, covalently coupled to phycoerythrin-labeled latex microspheres, and the other by the same DHT derivative, coupled to a fluorescein derivative (4).