A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR ␣ subtypes from spotted seatrout A LTHOUGH THE IMPORTANCE of rapid (i.e. nonclassical) steroid actions initiated at the cell surface through binding to steroid membrane receptors has become more widely accepted within the past few years, details of the initial steroid-mediated events, including the identities of the steroid membrane receptors and their mechanisms of action, remain unclear and are surrounded by controversy (1-3). There is clear evidence that a variety of receptor proteins are involved in initiating these nonclassical steroid actions in different cell models, including nuclear steroid receptors or nuclear steroid receptor-like forms (1, 2, 4), receptors for other ligands that also bind steroids (2, 5), and unidentified receptors with different characteristics from those of any known receptors (2, 6). Recently, a novel cDNA was discovered in spotted seatrout ovaries that has several characteristics of the progestin membrane receptor (mPR) mediating progestin induction of oocyte maturation in this species by a nongenomic mechanism (7). The seatrout cDNA (st-mPR␣) encodes a 40 kDa protein, which has seven transmembrane domains, and receptor activation alters pertussis toxin-sensitive adenylyl cyclase activity, both of which suggest stmPR␣ is a G protein-coupled receptor (GPCR) or GPCR-like protein (7). More than 20 closely related genes have been cloned from other vertebrate species, including three mPR subtypes in humans, named ␣, ␤, and ␥, which show high levels of expression in human reproductive, brain, and kidney tissues, respectively (8). The identification of a new class of putative steroid receptors, unrelated to nuclear steroid receptors, but instead related to GPCRs, provides a plausible explanation of how steroids can initiate rapid hormonal responses in target cells by activating receptors on the cell surface. There has been broad recognition of the potential significance of these findings (1, 9, 10) and also an extensive research effort to determine the distribution, hormonal regulation, and biological roles of the mPRs in various vertebrate models (11-16). However, critical information is still lacking on several key features of mPRs essential for clearly establishing this proposed alternative model of steroid action and for understanding its likely evolutionary origins.The st-mPR␣ protein has been localized to the plasma membrane of seatrout oocytes (7), but progestin binding and activation of signal transduction pathways in the plasma membranes of cells transfected with the st-mPR␣ and human mPRs remain to be demonstrated. To date, progestin binding has only First Published Online November 9, 2006Abbreviations: GPCR, G protein-coupled receptor; HLY3, hemolysin 3; hu-mPR␣, human membrane progestin receptor ␣; MMD, monocyte to macrophage differentiation protein; mPR, membrane progestin rece...

The polar molecular surface area is a dominating determinant for oral absorption and brain penetration of drugs that are transported by the transcellular route. This property should be considered in the early phase of drug screening.

The past decade has witnessed a paradigm shift in preclinical drug discovery with structure-based drug design (SBDD) making a comeback while high-throughput screening (HTS) methods have continued to generate disappointing results. There is a deficit of information between identified hits and the many criteria that must be fulfilled in parallel to convert them into preclinical candidates that have a real chance to become a drug. This gap can be bridged by investigating the interactions between the ligands and their receptors. Accurate calculations of the free energy of binding are still elusive; however progresses were made with respect to how one may deal with the versatile role of water. A corpus of knowledge combining X-ray structures, bioinformatics and molecular modeling techniques now allows drug designers to routinely produce receptor homology models of increasing quality. These models serve as a basis to establish and validate efficient rationales used to tailor and/or screen virtual libraries with enhanced chances of obtaining hits. Many case reports of successful SBDD show how synergy can be gained from the combined use of several techniques. The role of SBDD with respect to two different classes of widely investigated pharmaceutical targets: (a) protein kinases (PK) and (b) G-protein coupled receptors (GPCR) is discussed. Throughout these examples prototypical situations covering the current possibilities and limitations of SBDD are presented.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) activate the LH receptor/cyclic AMP (cAMP) signaling pathway to induce ovulation. As an alternative to parenterally administered hCG to treat anovulatory infertility, orally active low molecular weight (LMW) LHR agonists have been developed at Organon. In this paper, we present the mechanism of action of a prototypic, nanomolar potent and almost full LHR agonist, Org 43553. Org 43553 interacts with the endodomain of the LHR, whereas LH acts via the N-terminal exodomain. LH stimulates the cAMP pathway with an EC50 of 35 pM, but this stimulation is not antagonized by simultaneous incubation with Org 43553. At nanomolar concentrations, LH also stimulates phospholipase C (PLC), but Org 43553 is hardly able to do so. In contrast, Org 43553 inhibits LH-induced PLC (IC50 approximately 10 nM). While Org 43553 stimulates dissociation of [125I]hCG from the LHR and reduces [125I]hCG binding, LH reduces specific [3H]Org 43553 binding. We conclude that Org 43553 is a signaling-selective, allosteric LHR agonist. We hypothesize that Org 43553 and LH induce a similar LHR conformation necessary for activating adenylyl cyclase, which initiates most, if not all, physiological responses of LH.

Recent results showing that the binding characteristics of 33 steroids for human membrane progesterone receptor alpha (hu-mPRα) differ from those for the nuclear progesterone receptor (nPR) suggest that hu-mPRα-specific agonists can be identified for investigating its physiological functions. The binding affinities of an additional 21 steroids for hu-mPRα were determined to explore the structure-activity relationships in more detail and to identify potent, specific mPRα agonists. Four synthetic progesterone derivatives with methyl or methylene groups on positions 18 or 19, 18a-methylprogesterone (18-CH 3 P4, Org OE 64-0), 13-ethenyl-18-norprogesterone (18-CH 2 P4, Org 33663-0), 19a-methylprogesterone (19-CH 3 P4, Org OD 13-0) and 10-ethenyl-19-norprogesterone (19-CH 2 P4, Org OD 02-0), showed similar or higher affinities than progesterone for hu-mPRα and displayed mPRα agonist activities in G-protein and MAP kinase activation assays. All four steroids also bound to the nPR in cytosolic fractions of MCF-7 cells. However, two compounds, 19-CH 2 P4 and 19-CH 3 P4, showed no nPR agonist activity in a nPR reporter assay and therefore are selective mPRα agonists suitable for physiological investigations. The structure-binding relationships of the combined series of 54 steroids for hu-mPRα deviated strikingly from those of a published set of 60 3-keto or 3-desoxy steroids for nPR. Close correlations were observed between the receptor binding affinities of the steroids and their physicochemical properties calculated by comparative molecular field analysis (CoMFA) for both hu-mPRα and nPR. A comparison of the CoMFA field graphs for the two receptors revealed several differences in the structural features required for binding to hu-mPRα and nPR which could be exploited to develop additional mPR-specific ligands.