Purification and characterization of recombinant human renin for X-ray crystallization studies

<p>Abstract</p> <p>Background</p> <p>The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies.</p> <p>Results</p> <p>A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained.</p> <p>Conclusion</p> <p>This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.</p

Nasal aerodynamics protects brain and lung from inhaled dust in subterranean diggers Ellobius talpinus

Inhalation of air-dispersed sub-micrometre and nano-sized particles presents a risk factor for animal and human health. Here, we show that nasal aerodynamics plays a pivotal role in the protection of the subterranean mole vole Ellobius talpinus from an increased exposure to nano-aerosols. Quantitative simulation of particle flow has shown that their deposition on the total surface of the nasal cavity is higher in the mole vole than in a terrestrial rodent Mus musculus (mouse), but lower on the olfactory epithelium. In agreement with simulation results, we found a reduced accumulation of manganese in olfactory bulbs of mole voles in comparison with mice after the inhalation of nano-sized MnCl(2) aerosols. We ruled out the possibility that this reduction is owing to a lower transportation from epithelium to brain in the mole vole as intranasal instillations of MnCl(2) solution and hydrated nanoparticles of manganese oxide MnO · (H(2)O)(x) revealed similar uptake rates for both species. Together, we conclude that nasal geometry contributes to the protection of brain and lung from accumulation of air-dispersed particles in mole voles

Discovery of a Novel, Orally Efficacious Liver X Receptor (LXR) β Agonist

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor β (LXRβ) agonist (<b>17</b>). Contour technology is a structure-based drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRβ and a docked 2-(methylsulfonyl)­benzyl alcohol fragment (<b>6</b>), Contour was used to design agonists containing a piperazine core. Compound <b>17</b> binds to LXRβ with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes <i>in vitro</i> and <i>in vivo</i>. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis