ABSTRACT
Synthetic progesterone and 5α/ß-pregnane-3,20-dione derivatives were evaluated as in vitro and in vivo modulators of multidrug-resistance (MDR) using two P-gp-expressing human cell lines, the non-steroidogenic K562/R7 erythroleukaemia cells and the steroidogenic NCI-H295R adrenocortical carcinoma cells, both resistant to doxorubicin. The maximal effect in both cell lines was observed for 7α-O-benzoyloxy,11α(R)-O-tetrahydropyranyloxy-5ß-pregnane-3,20-dione 4. This modulator co-injected with doxorubicin significantly decreased the tumour size and increased the survival time of immunodeficient mice xenografted with NCI-H295R or K562/R7 cells.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Pregnanes/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Mice , Mice, SCID , Molecular Conformation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Pregnanes/chemical synthesis , Pregnanes/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Binding studies of the interaction of immobilized 1alpha- and 17alpha-aminoalkyl derivatives of 5alpha-dihydrotestosterone (DHT) with purified N-deglycosylated homodimeric human sex hormone-binding globulin (SHBG) were performed using a surface plasmon resonance biosensor. These 1alpha- and 17alpha-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(a)1) for the binding of these immobilized DHT derivatives to the first binding site of SHBG, determined by SPR measurements, were 0.16 x 10(7) M(-1) for 17alpha-aminopropyl-17beta-hydroxy-5alpha-androstan-3-one (1), 1.64 x 10(7) M(-1) for 17alpha-aminocaproyl-17beta-hydroxy-5alpha-androstan-3-one (2), and 1.2 x 10(8) M(-1) for 1alpha-aminohexyl-17beta-hydroxy-5alpha-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 x 10(7) M(-1) for 1, 1.50 x 10(7) M(-1) for 2, and 140 x 10(7) 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 1alpha-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).
Subject(s)
Dihydrotestosterone/analogs & derivatives , Dihydrotestosterone/metabolism , Sex Hormone-Binding Globulin/metabolism , Binding Sites , Biosensing Techniques/methods , Dimerization , Flow Cytometry , Humans , Kinetics , Latex , Microspheres , Models, Chemical , Protein Binding , Surface Plasmon Resonance/methods , Testosterone/metabolism , ThermodynamicsABSTRACT
We report on the syntheses of 1 alpha- and 17 alpha-aminoalkyl dihydrotestosterone (DHT) derivatives and the particularly high binding affinity of the 1 alpha-aminohexyl ligand for human sex hormone-binding globulin (SHBG). The two 17 alpha-aminopropyl-17 beta-hydroxy-5 alpha-androstan-3-one (1) and 17 alpha-aminocaproylamidoethyl-17 beta-hydroxy-5 alpha-androstan-3-one (2) derivatives were synthesized via a 17beta-spirooxirane intermediate in high yields. The 1 alpha-aminohexyl-17 beta-hydroxy-5 alpha-androstan-3-one compound (3) was obtained in a seven step synthesis using a copper-catalyzed conjugate addition of a omega-silyloxyhexyl Grignard reagent to 17 beta-benzoyloxy-5 alpha-androst-1-en-3-one. All structures were elucidated based on 1H NMR spectroscopy and mass spectral analyses. The three aminosteroid derivatives were tested as ligands for SHBG by competition experiments with tritiated testosterone as tracer under equilibrium conditions. The association constants of the two 17 alpha-DHT derivatives were approximately 1 x 10(7) M(-1), whereas the 1 alpha-DHT derivative showed a remarkably high binding affinity to SHBG with an association constant of 1.40 x 10(9) M(-1). These aminoalkyl derivatives, substituted either at the D-ring or the A-ring of the steroid skeleton, can be easily coupled onto a carboxymethylated solid state surface of a biosensor. Such a device lends itself to kinetic and thermodynamic studies aimed to provide a better understanding of the biospecific interaction of steroids with SHBG.
