A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8

Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity. The data analysis revealed that the presence of heparin affects all steps of the enzyme reaction: (i) it decreases 3.5-fold the k1 and 4.0-fold the k−1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k2 (2.7-fold), and also decrease in k3 (3.5-fold). The large values of ΔG = 12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys25)-S−/(His163)-Im+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme. Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface

A “Genome-to-Lead” Approach for Insecticide Discovery: Pharmacological Characterization and Screening of <em>Aedes aegypti</em> D₁-like Dopamine Receptors

<div><h3>Background</h3><p>Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides.</p> <h3>Methodology/Principal Findings</h3><p>We describe a “genome-to-lead” approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (<em>Aa</em>DOP1 and <em>Aa</em>DOP2) from the yellow fever mosquito, <em>Aedes aegypti</em>. Sequence analyses indicated that these receptors are orthologous to arthropod D₁-like (Gα_s-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of <em>Aa</em>DOP1 and <em>Aa</em>DOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC₅₀: <em>Aa</em>DOP1 = 3.1±1.1 nM; <em>Aa</em>DOP2 = 240±16 nM). Interestingly, only <em>Aa</em>DOP1 exhibited sensitivity to epinephrine (EC₅₀ = 5.8±1.5 nM) and norepinephrine (EC₅₀ = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at <em>Aa</em>DOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at <em>Aa</em>DOP2. <em>In vitro</em> comparison studies between <em>Aa</em>DOP2 and the human D₁ dopamine receptor (hD₁) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as <em>Aa</em>DOP2-selective compounds. In subsequent <em>Ae. aegypti</em> larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery.</p> <h3>Conclusions/Significance</h3><p>This research provides a “proof-of-concept” for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with <em>in vivo</em> toxicity toward mosquitoes.</p> </div

Lumican Binds ALK5 to Promote Epithelium Wound Healing

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC(13) peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo

Confirmation and secondary assays for “hit” antagonists of <i>Aa</i>DOP2 and human D₁ receptor.

<p>Select chemistries and the assay control (SCH23390) were tested in dose-response cAMP assays in the presence of 3 µM dopamine in <i>Aa</i>DOP2- or 100 nM dopamine in hD₁-expressing cells (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd-0001478-g005" target="_blank">Figure 5</a>). Compounds with IC₅₀ values ≥10 µM are considered to lack activity at <i>Aa</i>DOP2 and were not tested at hD₁. N.D. = not determined; hD₁ = Human D₁ dopamine receptor.</p

Neighbor-joining sequence analysis of <i>Aedes aegypti Aa</i>DOP1 and AaDOP2 and representative biogenic amine receptors.

<p>The deduced amino acid sequences for the mosquito dopamine receptors <i>Aa</i>DOP1 and AaDOP2 and additional receptors for dopamine, muscarinic acetylcholine, octopamine, serotonin, and tyramine from <i>Drosophila melanogaster</i> and <i>Apis mellifera</i>, as well as the human D₁-like and D₂-like dopamine receptors were aligned for use in the analysis. Bootstrap values (100 replicates) are indicated with numbers at supported branches. The outgroup is a <i>D. melanogaster</i> diuretic hormone receptor, a Class B GPCR. Abbreviations: <i>Aa</i> = <i>Ae. aegypti</i>; <i>Is</i> = <i>I. scapularis</i>; <i>Dm</i> = <i>D. melanogaster</i>; <i>Am</i> = <i>A. mellifera</i>; <i>Hs</i> = <i>H. sapiens</i>. Sequences: <i>Is</i>dop1, D₁-like dopamine receptor (ISCW001496); <i>Is</i>dop2, D₁-like dopamine receptor (ISCW008775); <i>Dm</i>D-Dop1, D₁-like dopamine receptor (P41596); <i>Dm</i>DAMB, D₁-like dopamine receptor (DopR99B/DAMB: AAC47161), <i>Dm</i>DD2R, D₂-like dopamine receptor (DD2R-606: AAN15955); <i>Dm</i>Dih, diuretic hormone 44 receptor 1 (NP_610960.1); <i>Dm</i>mAChR, muscarinic acetylcholine receptor (AAA28676); <i>Dm</i>OAMB, octopamine receptor in mushroom bodies, isoform A (NP_732541); DM5HT1A, serotonin receptor 1A, isoform A (NP_476802); <i>Dm</i>Tyr, tyramine receptor (CG7431: NP_650652); <i>Am</i>DOP1, D₁-like dopamine receptor (dopamine receptor, D1, NP_001011595); <i>Am</i>DOP2, D₁-like dopamine receptor (dopamine receptor 2, NP_001011567), <i>Am</i>DOP3, D₂-like dopamine receptor (<i>Am</i>DOP3, NP_001014983); <i>Am</i>mAChR, muscarinic acetylcholine receptor (XP_395760); <i>Am</i>OA1, octopamine receptor (oar, NP_001011565); <i>Am</i>5HT1A, serotonin receptor (5ht-1, NP_001164579); <i>Am</i>Tyr, tyramine receptor (XP_394231); <i>Hs</i>D1, D₁-like dopamine receptor (D(1A), NP_000785); <i>Hs</i>D2,D₂-like dopamine receptor (D(2), NP_000786); <i>Hs</i>D3, D₂-like dopamine receptor (D(3), NP_000787); <i>Hs</i>D4, D₂-like dopamine receptor (D(4), NP_000788); <i>Hs</i>D5, D₁-like dopamine receptor (D(1B)/D5, NP_000789).</p