ABSTRACT
(1)H NMR Saturation Transfer Difference (STD) experiments were applied to study the binding of aspirin and of an anti-inflammatory complex of Cu(I), namely [Cu(tpp)(pmt)](2) [pmt = 2-mercaptopyrimidine), synthesized in an attempt to develop novel metallotherapeutic molecules. While aspirin showed only very weak binding, the complex [Cu(tpp)(pmt)](2) clearly favored binding to LOX-1. In silico docking experiments in LOX-1 showed that aspirin does only weakly bind to LOX-1, while the complex binds with high affinity. In addition, docking experiments and molecular dynamics (MD) simulations showed that the complex binds via hydrogen bonding (HB), to an allosteric site of LOX-1, revealing that this enzyme has more than one accessible site for complex metallotherapeutic molecules. When aspirin was added in the solution containing LOX and the complex [Cu(tpp)(pmt)](2), the former was shown to hinder the binding of the Cu complex significantly. This may be interpreted as the copper complex aiding the transfer of aspirin through an acid-base reaction at the LOX enzyme which subsequently blocks its binding.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Aspirin/metabolism , Catalytic Domain , Copper/chemistry , Lipoxygenase/chemistry , Lipoxygenase/metabolism , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein BindingABSTRACT
The two new synthetic analogues of the MBP(83-99) epitope substituted at Lys(91) (primary TCR contact) with Phe [MBP(83-99) (Phe(91))] or Tyr [MBP(83-99) (Tyr(91))], have been structurally elucidated using 1D and 2D high resolution NMR studies. The conformational analysis of the two altered peptide ligands (APLs) has been performed and showed that they adopt a linear and extended conformation which is in agreement with the structural requirements of the peptides that interact with the HLA-DR2 and TCR receptors. In addition, Molecular Dynamics (MD) simulations of the two analogues in complex with HLA-DR2 (DRA, DRB1*1501) and TCR were performed. Similarities and differences of the binding motif of the two analogues were observed which provide a possible explanation of their biological activity. Their differences in the binding mode in comparison with the MBP(83-99) epitope may also explain their antagonistic versus agonistic activity. The obtained results clearly indicate that substitutions in crucial amino acids (TCR contacts) in combination with the specific conformational characteristics of the MBP(83-99) immunodominant epitope lead to an alteration of their biological activity. These results make the rational drug design intriguing since the biological activity is very sensitive to the substitution and conformation of the mutated MBP epitopes.
Subject(s)
Epitopes/chemistry , HLA-DR2 Antigen/immunology , Myelin Basic Protein/chemistry , Myelin Basic Protein/immunology , Peptide Fragments/chemistry , Receptors, Antigen, T-Cell/immunology , Amino Acid Sequence , Epitopes/immunology , Humans , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Molecular Sequence Data , Peptide Fragments/immunology , Protein ConformationABSTRACT
Theoretical and structural studies followed by the directed synthesis and in vitro biological tests lead us to novel noncovalent thrombin pseudopeptide inhibitors. We have incorporated an azapeptide scaffold into the central part of the classical tripeptide D-Phe-Pro-Arg inhibitor structure thus eliminating one stereogenic center from the molecule. A series of compounds has been designed to optimize the occupancy of the S2 pocket of thrombin. Increased hydrophobicity at P2 provides an enhanced fit into this active site S2 pocket. In the present paper, we also report on the structure of these inhibitors in solution and conformational analysis of inhibitors in the active site in order to asses the consequences of the replacement of the central alpha-CH by a nitrogen functionality. In vitro biological testing of the designed inhibitors shows that elimination of R, S stereoisomerism and restriction of conformational freedom influences the binding of inhibitors in a favorable fashion.
Subject(s)
Antithrombins , Aza Compounds/chemical synthesis , Enzyme Inhibitors , Oligopeptides/chemistry , Phenylalanine , Antithrombins/chemical synthesis , Antithrombins/chemistry , Antithrombins/pharmacokinetics , Antithrombins/pharmacology , Aza Compounds/chemistry , Aza Compounds/pharmacokinetics , Aza Compounds/pharmacology , Binding Sites , Catalysis , Combinatorial Chemistry Techniques/methods , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Molecular Mimicry , Molecular Structure , Nipecotic Acids/pharmacology , Oligopeptides/pharmacology , Phenylalanine/analogs & derivatives , Phenylalanine/chemical synthesis , Phenylalanine/chemistry , Phenylalanine/pharmacokinetics , Phenylalanine/pharmacology , Serine/analogs & derivatives , Serine/pharmacology , Spectrophotometry, Infrared , Spectroscopy, Fourier Transform Infrared , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The novel amide linked Angiotensin II (ANG II) cyclic analogue cyclo(3, 5) -[Sar(1)-Lys(3)-Glu(5)-Ile(8)] ANG II (18) has been designed, synthesized and bioassayed in anesthetized rabbits. The constrained cyclic analogue with a lactam amide bridge linking a Lys-Glu pair at positions 3 and 5 and possessing Ile at position 8, was synthesized by solution procedure using the maximum protection strategy. This analogue was found to be inhibitor of Angiotensin II. NMR spectroscopy coupled with computational analysis showed clustering between the side chains of the key aminoacids Tyr(4)-His(6)-Ile(8) similar to that observed with ANG II. The obtained data show that only pi*--pi* interactions observed in ANG II or its superagonist Sar(1) [ANG II] are missing. Therefore, it can be concluded that these interactions are essential for agonist activity. Conformational analysis comparisons between AT(1) antagonists losartan, eprosartan and irbesartan with C-terminal segment of cyclic compound 18 revealed structural similarities.
Subject(s)
Angiotensin II/antagonists & inhibitors , Angiotensin II/chemistry , Angiotensin II/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Thiophenes , Acrylates/chemistry , Acrylates/pharmacology , Angiotensin II/analogs & derivatives , Angiotensin II/chemical synthesis , Animals , Antihypertensive Agents/chemistry , Antihypertensive Agents/pharmacology , Biochemistry/methods , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Drug Design , Imidazoles/chemistry , Imidazoles/pharmacology , Irbesartan , Losartan/chemistry , Losartan/pharmacology , Magnetic Resonance Spectroscopy , Male , Models, Molecular , Peptides, Cyclic/chemical synthesis , Rabbits , Structure-Activity Relationship , Tetrazoles/chemistry , Tetrazoles/pharmacologyABSTRACT
The IICBGlc subunit of the Escherichia coli glucose transporter consists of two domains, the membrane-spanning IIC domain, and the hydrophilic IIB domain which contains the phosphorylation site (Cys421). A functional form of the IIB domain was over-expressed separately and isotopically labelled with 13C and 15N. A variety of 15N-edited and 13C, 15N triple-resonance NMR experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on the evaluation of conformationally sensitive parameters including NOE effects, scalar couplings and chemical shifts, the secondary structure of the IIB domain is presented. The protein is comprised of four beta-strands forming an antiparallel beta-sheet, two larger alpha-helices at the N- and C-termini and a smaller helical structure of residues 52-58.