Structure of a Bovine Thrombin-Hirudin\u3csub\u3e51-65\u3c/sub\u3e Complex Determined by a Combination of Molecular Replacement and Graphics. Incorporation of Known Structural Information in Molecular Replacement

Crystals of the bovine thrombin-hirudins51-65 complex have space group P6122 with cell constants a = 116.4, and c = 200.6 Å and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 Å. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is -thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement

Structure of a Bovine Thrombin-Hirudin\u3csub\u3e51-65\u3c/sub\u3e Complex Determined by a Combination of Molecular Replacement and Graphics. Incorporation of Known Structural Information in Molecular Replacement

Crystals of the bovine thrombin-hirudins51-65 complex have space group P6122 with cell constants a = 116.4, and c = 200.6 Å and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 Å. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is -thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement

Comparison of the Antinociceptive and Antirewarding Profiles of Novel Bifunctional Nociceptin Receptor/μ-Opioid Receptor Ligands: Implications for Therapeutic Applications

The nociceptin receptor (NOPr), a member of the opioid receptor family, is a target for the treatment of pain and drug abuse. Nociceptin/orphanin FQ (N/OFQ), the endogenous peptide for NOPr, not only modulates opioid antinociception, but also blocks the rewarding effects of several abused drugs, such as morphine, cocaine, and amphetamine. We hypothesized that NOPr agonists, with bifunctional activity at the μ-opioid receptor (MOPr), may function as nonaddicting analgesics or as drug abuse medications. Bifunctional small-molecule NOPr agonists possessing different selectivities and efficacies at MOPr were evaluated in an acute thermal antinociception assay, and for their ability to induce conditioned place preference (CPP) and their effect on morphine-induced CPP. 1-(1-Cyclooctylpiperidin-4-yl)-indolin-2-one) (SR14150), a high-affinity NOPr partial agonist, with low MOPr affinity and efficacy, produced analgesia that was naloxone-reversible. SR14150 did not induce CPP alone, nor did it attenuate morphine-induced CPP. 3-Ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), which has high affinity for both NOPr and MOPr, full agonist activity at NOPr, and partial agonist activity at MOPr, was also a potent analgesic and produced CPP alone, but also modestly attenuated morphine CPP. 1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidinl-4-yl)-indolin-2-one (SR16835), a NOPr full agonist and low-affinity MOPr partial agonist, was not antinociceptive, did not produce CPP alone, but attenuated morphine CPP. Our results suggest that NOPr full-agonist activity is required to modulate opioid-induced reward, whereas a bifunctional NOPr/MOPr partial agonist profile may be suitable as a nonaddicting analgesic. The opioid-modulating effects of the NOPr ligands may be used effectively to produce better medications for treatment of drug abuse and pain

Tailored magnetic fields in the melt extraction of metallic filaments

Porous bodies that are resistant to corrosion at high temperatures and thermal shock may be produced from metallic fibers. In order to accomplish reasonable homogeneity and high porosity, the cross-sectional area of the fibers and the width of distribution thereof need to be small. This article studies two techniques for making fibers. Melt extraction out of a crucible yields filaments with a typical diameter ranging from 50 to 200 mu m, which is too thick. Also patented for a long time is the extraction from a pendant drop. Even though relatively fine fibers can be manufactured with this method, it never exceeded crucible extraction with respect to industrial importance owing to the low productivity of the process. The present article addresses the drawbacks of both variants of melt extraction of metallic filaments. Because metallic melts are electrically conducting, the use of magnetic fields allows for contactless process optimization. It is well believed that increasing the extraction speed diminishes the fiber diameter. Being not always true, at least in the case of crucible melt extraction, as indicated by the present findings, however, undesired fluid flow, i.e., turbulence, imposes an upper limit on the rotation rate of the extraction wheel. Application of a static magnetic field leads to both higher wheel speed and thinner filaments. The low productivity of extraction from the molten tip of a rod suffers from the fact that only one melt drawing edge can be used. As the bare rod is problematic with respect to heating its tip in contact with the extraction wheel, it is challenging to melt the entire edge of a sheet. A special design of the induction-heating magnetic field is also proposed to solve also this task

Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): Ligand-based analysis of structural factors influencing intrinsic activity at NOP

The recently discovered fourth member of the opioid receptor family, the nociceptin receptor (NOP) and its endogenous ligand, the heptadecaptide nociceptin, are involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small-molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents a brief overview of the several recently reported NOP ligands, classified as NOP agonists and antagonists, with an emphasis on the analysis of the structural features that may be important for modulating the agonist/antagonist profile (intrinsic activity) of these ligands. Structure-activity relationships in our own series of dihydroindolinone-based NOP ligands and those of the various reported ligands indicate that the lipophilic substituent on the common basic nitrogen present in all NOP ligands plays a role in determining the agonist/antagonist profile of the NOP ligand. This analysis provides a basis for the rational drug design of NOP ligands of desired intrinsic activity and provides a framework for developing pharmacophore models for high affinity binding and intrinsic activity at the NOP receptor. Since NOP agonists and antagonists both have therapeutic value, rational approaches for obtaining both within a high-affinity binding class of compounds are very useful for designing potent and selective NOP ligands with the desired profile of intrinsic efficacy