1.Exploring the interaction of N/S compounds with a dicopper center: tyrosinase inhibition and model studies.
Buitrago E1, Vuillamy A, Boumendjel A, Yi W, Gellon G, Hardré R, Philouze C, Serratrice G, Jamet H, Réglier M, Belle C. Inorg Chem. 2014 Dec 15;53(24):12848-58. doi: 10.1021/ic501829s. Epub 2014 Nov 21.
Tyrosinase (Ty) is a copper-containing enzyme widely present in plants, bacteria, and humans, where it is involved in biosynthesis of melanin-type pigments. Development of Ty inhibitors is an important approach to control the production and the accumulation of pigments in living systems. In this paper, we focused our interest in phenylthiourea (PTU) and phenylmethylene thiosemicarbazone (PTSC) recognized as inhibitors of tyrosinase by combining enzymatic studies and coordination chemistry methods. Both are efficient inhibitors of mushroom tyrosinase and they can be considered mainly as competitive inhibitors. Computational studies verify that PTSC and PTU inhibitors interact with the metal center of the active site. The KIC value of 0.93 μM confirms that PTSC is a much more efficient inhibitor than PTU, for which a KIC value of 58 μM was determined. The estimation of the binding free energies inhibitors/Ty confirms the high inhibitor efficiency of PTSC.
2.Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon Beta-1a: potential implications for protein aggregation and immunogenicity.
Torosantucci R1, Sharov VS, van Beers M, Brinks V, Schöneich C, Jiskoot W. Mol Pharm. 2013 Jun 3;10(6):2311-22. doi: 10.1021/mp300665u. Epub 2013 May 2.
Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products.
3.Human neutrophil-pulmonary microvascular endothelial cell interactions in vitro: differential effects of nitric oxide vs. peroxynitrite.
Shelton JL1, Wang L, Cepinskas G, Inculet R, Mehta S. Microvasc Res. 2008 Aug;76(2):80-8. doi: 10.1016/j.mvr.2008.06.001. Epub 2008 Jun 14.
Sepsis-induced acute lung injury is characterized by activation and injury of pulmonary microvascular endothelial cells (PMVEC), increased neutrophil-PMVEC adhesion and migration, and trans-PMVEC high-protein edema. Inducible NO synthase (iNOS) inhibits septic murine neutrophil migration in vivo and in vitro. The effects of NO in human neutrophil-PMVEC interactions are not known. We isolated human PMVEC using magnetic bead-bound anti-PECAM antibody. Confluent PMVEC at passage 3-4 were co-cultured with human neutrophils for assessment of neutrophil-PMVEC adhesion, and trans-PMVEC neutrophil migration and Evans-Blue dye-labeled albumin leak. Two NO donors (spermine-NONOate, S-nitroso-N-acetylpenicillamine) attenuated both cytomix-enhanced neutrophil-PMVEC adhesion by 64+/-14% (p<0.01) and 32+/-3% (p<0.05), respectively, and cytomix-induced trans-PMVEC neutrophil migration by 85+/-16% (p<0.01) and 43+/-5% (p<0.01), respectively.
4.The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent.
Lussenburg BM1, Keizers PH, de Graaf C, Hidestrand M, Ingelman-Sundberg M, Vermeulen NP, Commandeur JN. Biochem Pharmacol. 2005 Oct 15;70(8):1253-61.
The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1'-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity.
