Improving the permeability of the hydroxyethylamine BACE-1 inhibitors: Structure–activity relationship of P2′ substituents

Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2′ position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.     

Fragment screening of inhibitors for MIF tautomerase reveals a cryptic surface binding site

In the course of a fragment screening campaign by in silico docking followed by X-ray crystallography, a novel binding site for migration inhibitory factor (MIF) inhibitors was demonstrated. The site is formed by rotation of the side-chain of Tyr-36 to reveal a surface binding site in MIF that is hydrophobic and surrounded by aromatic side-chain residues. The crystal structures of two small inhibitors that bind to this site and of a quinolinone inhibitor, that spans the canonical deep pocket near Pro-1 and the new surface binding site, have been solved. These results suggest new opportunities for structure-based design of MIF inhibitors.     

Halogenation of 4-hydroxy/amino-3-methoxyphenyl acetamide TRPV1 agonists showed enhanced antagonism to capsaicin

As an extension of our analysis of the effect of halogenation on thiourea TRPV1 agonists, we have now modified selected 4-hydroxy(or 4-amino)-3-methoxyphenyl acetamide TRPV1 agonists by 5- or 6-halogenation on the aromatic A-region and evaluated them for potency for TRPV1 binding and regulation and for their pattern of agonism/antagonism (efficacy). Halogenation shifted the functional activity at TRPV1 toward antagonism with a greater extent of antagonism as the size of the halogen increased (I>Br>Cl), as previously observed for the thiourea series. The extent of antagonism was greater for halogenation at the 5-position than at the 6-position, in contrast to SAR for the thiourea series. In this series, compounds 55 and 75 showed the most potent antagonism, with K(i) (ant)=2.77 and 2.19nM, respectively, on rTRPV1 expressed in Chinese hamster ovary cells. The compounds were thus ca. 40-60-fold more potent than 6'-iodononivamide.     

Low temperature dynamic mapping reveals unexpected order and disorder in troponin

Troponin is a pivotal regulatory protein that binds Ca(2+) reversibly to act as the muscle contraction on-off switch. To understand troponin function, the dynamic behavior of the Ca(2+)-saturated cardiac troponin core domain was mapped in detail at 10 °C, using H/D exchange-mass spectrometry. The low temperature conditions of the present study greatly enhanced the dynamic map compared with previous work. Approximately 70% of assessable peptide bond hydrogens were protected from exchange sufficiently for dynamic measurement. This allowed the first characterization by this method of many regions of regulatory importance. Most of the TnI COOH terminus was protected from H/D exchange, implying an intrinsically folded structure. This region is critical to the troponin inhibitory function and has been implicated in thin filament activation. Other new findings include unprotected behavior, suggesting high mobility, for the residues linking the two domains of TnC, as well as for the inhibitory peptide residues preceding the TnI switch helix. These data indicate that, in solution, the regulatory subdomain of cardiac troponin is mobile relative to the remainder of troponin. Relatively dynamic properties were observed for the interacting TnI switch helix and TnC NH(2)-domain, contrasting with stable, highly protected properties for the interacting TnI helix 1 and TnC COOH-domain. Overall, exchange protection via protein folding was relatively weak or for a majority of peptide bond hydrogens. Several regions of TnT and TnI were unfolded even at low temperature, suggesting intrinsic disorder. Finally, change in temperature prominently altered local folding stability, suggesting that troponin is an unusually mobile protein under physiological conditions.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Quantification of cyanuric acid residue in human urine using high performance liquid chromatography–tandem mass spectrometry

Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography–tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00–200 ng/mL. The limit of detection was 0.60 ng/mL and the relative standard deviations were 2.8–10.5% across the calibration range. The relative recovery of cyanuric acid was 100–104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.     

Microdissection of the gene expression codes driving nephrogenesis

The kidney represents an excellent model system for learning the principles of organogenesis. It is intermediate in complexity, and employs many commonly used developmental processes. As such, kidney development has been the subject of intensive study, using a variety of techniques, including in situ hybridization, organ culture and gene targeting, revealing many critical genes and pathways. Nevertheless, proper organogenesis requires precise patterns of cell type specific differential gene expression, involving very large numbers of genes. This review is focused on the use of global profiling technologies to create an atlas of gene expression codes driving development of different mammalian kidney compartments. Such an atlas allows one to select a gene of interest, and to determine its expression level in each element of the developing kidney, or to select a structure of interest, such as the renal vesicle, and to examine its complete gene expression state. Novel component specific molecular markers are identified, and the changing waves of gene expression that drive nephrogenesis are defined. As the tools continue to improve for the purification of specific cell types and expression profiling of even individual cells it is possible to predict an atlas of gene expression during kidney development that extends to single cell resolution.     

The dopamine metabolite 3-methoxytyramine is a neuromodulator

Dopamine (3-hydroxytyramine) is a well-known catecholamine neurotransmitter involved in multiple physiological functions including movement control. Here we report that the major extracellular metabolite of dopamine, 3-methoxytyramine (3-MT), can induce behavioral effects in a dopamine-independent manner and these effects are partially mediated by the trace amine associated receptor 1 (TAAR1). Unbiased in vivo screening of putative trace amine receptor ligands for potential effects on the movement control revealed that 3-MT infused in the brain is able to induce a complex set of abnormal involuntary movements in mice acutely depleted of dopamine. In normal mice, the central administration of 3-MT caused a temporary mild hyperactivity with a concomitant set of abnormal movements. Furthermore, 3-MT induced significant ERK and CREB phosphorylation in the mouse striatum, signaling events generally related to PKA-mediated cAMP accumulation. In mice lacking TAAR1, both behavioral and signaling effects of 3-MT were partially attenuated, consistent with the ability of 3-MT to activate TAAR1 receptors and cause cAMP accumulation as well as ERK and CREB phosphorylation in cellular assays. Thus, 3-MT is not just an inactive metabolite of DA, but a novel neuromodulator that in certain situations may be involved in movement control. Further characterization of the physiological functions mediated by 3-MT may advance understanding of the pathophysiology and pharmacology of brain disorders involving abnormal dopaminergic transmission, such as Parkinson's disease, dyskinesia and schizophrenia.     

Endothelial Cells Are Essential for the Self-Renewal and Repopulation of Notch-Dependent Hematopoietic Stem Cells

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Highlights? Coculture with endothelial cells promotes HSC expansion and self-renewal ? Endothelial cell effect on HSCs mediated by Notch signaling ? In vivo, sinusoidal endothelial cells (SECs) express Notch ligands ? Blocking vessel remodeling impairs hematopoietic recovery after irradiation