AbstractHemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (KD = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Synthesis and structure-activity relationships of tricyclic alpha-ethoxy-phenylpropionic acid derivatives guided by in vitro PPARalpha and PPARgamma transactivation data and computer modeling led to the identification of the novel carbazole analogue, 3q, with dual PPARalpha (EC(50) = 0.36 microM) and PPARgamma (EC(50) = 0.17 microM) activity in vitro. Ten days treatment of db/db mice with 3q improved the insulin sensitivity, as measured by OGTT, better than that seen with both pioglitazone and rosiglitazone treatment, suggesting in vivo PPARgamma activity. Likewise, 3q lowered plasma triglycerides and cholesterol in high cholesterol fed rats after 4 days treatment, indicating in vivo PPARalpha activity. Investigations of the pharmacokinetics of selected compounds suggested that extended drug exposure improved the in vivo activity of in vitro active compounds.

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5 Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Prolactin (PRL)4 is a protein hormone secreted by the anterior pituitary in vertebrates and possesses physiological functions of remarkable diversity, including effects on reproduction, lactation, and growth. PRL belongs to a family of homologous proteins comprising PRL, growth hormone (GH), and placental lactogen (PL). The biological effects associated with this cytokine family are mediated by two distinct classes of cell surface receptors, the PRL receptors (PRLR) and the GH receptors (GHR). The PRL/PL/GH biology is governed by a delicate balance between receptor cross-reactivity and selectivity; PRL and PL bind selectively to PRLR, whereas GH is capable of binding both PRLR and GHR.After being proposed more than a decade ago (1), hormoneinduced receptor dimerization became generally accepted as the model for cytokine receptor activation. For the PRL family members, the model describes the signaling molecular entity as a ternary complex between one hormone molecule and a receptor homodimer assembled in a strictly sequential and hormonedependent fashion; first the hormone ligand engages via binding site 1 (BS1) in high affinity binding to one receptor chain forming a 1:1 hormone-receptor complex. This complex constitutes the template for binding a second, identical receptor molecule via binding site 2 (BS2), resulting in the active 1:2 complex. However, the model has been challenged by an increasing body of experimental evidence, initially reported for the homologous human erythropoietin receptor (2) and later for GHR (3) and PRLR (4). These studies suggest that preformed, inactive dimers exist in the absence of hormone. Thus, receptor dimerization is a necessary but not sufficient event for receptor activation and, notably, not strictly ligand-dependent. For both human erythropoietin receptor (5) and GHR (3), mechanistic models have been proposed, where receptor activation involves relative rotations and movements of receptor subunits induced by hormone binding. Allosteric reorganization of the intracellular receptor domains brings associated JAK2 kinases into close proximity, allowing their activation by cross-phosphorylation. This initial activation step triggers a cascade of molecular events leading to the functional receptor response (6).The...

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