Neurosteroids: 3 alpha-hydroxy-5 alpha-pregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats.

A RIA procedure for measuring progesterone (PROG), 5 alpha-pregnane-3,20-dione (5 alpha-DH PROG), and 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-TH PROG) has been developed and validated by GLC/mass spectrometry. Measurements were made in intact and adrenalectomized (ADX) male rats, in cyclic, pregnant, spayed, and spayed-ADX females, and in both males and spayed females injected with PROG. The predominant contribution of the ovary to the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain, was indicated by its larger levels in females, in particular during pregnancy, and by its presence in ovarian tissue and disappearance after ovariectomy. An additional adrenal origin in both males and females was shown. Neither PROG nor 5 alpha-DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy and gonadectomy, thus suggesting that PROG might be synthetized de novo in brain. However, the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain of female rats were positively correlated with the concentrations of PROG in plasma, indicating that plasma PROG was the major precursor of 3 alpha,5 alpha-TH PROG. The direct formation of 3 alpha,5 alpha-TH PROG from PROG in brain was strongly suggested by the increased 3 alpha,5 alpha-TH PROG/PROG ratios in brain vs. plasma, when measured in control females, and after injection of PROG to both males and OVX females. It was previously reported that 3 alpha,5 alpha-TH PROG is a sedative/anxiolytic steroid, as a result of its binding to gamma-aminobutyric acid (GABA)A receptors and allosteric potentiation of GABAcergic neurotransmission. Its concentrations in brain reach indeed the neuroactive range in cyclic and pregnant females, and are compatible with a physiological role of this neurosteroid.

Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance.

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.