ABSTRACT
The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.
Subject(s)
S100 Proteins/chemistry , Animals , Calcium/chemistry , Crystallography, X-Ray , Humans , Models, Molecular , S100 Proteins/genetics , S100 Proteins/isolation & purification , Solubility , Solutions , ZebrafishABSTRACT
Synthetic sulfonamide derivatives are a class of potent matrix metalloproteinase inhibitors (MMPI) that have potential for the treatment of diseases related to uncontrolled expression of these enzymes. The lack of selectivity of the large majority of such inhibitors, leading to the inhibition of MMPs in tissues other than the targeted one, has dramatically reduced the therapeutic interest in MMPIs. The recent development of efficient drug delivery systems that allow the transportation of a selected drug to its site of action has opened the way to new perspectives in the use of MMPIs. Here, a PAMAM-based divalent dendron with two sulfonamidic residues was synthesized. This nanomolar inhibitor binds to the catalytic domain of two MMPs as well as to the transmembrane human carbonic anhydrases (hCAs) XII, which is present in the eye and considered an antiglaucoma target. In the animal model of an experimental dry eye, no occurrence of dotted staining in eyes treated with our inhibitor was observed, indicating no symptoms of corneal desiccation.
Subject(s)
Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/pharmacology , Dry Eye Syndromes/drug therapy , Matrix Metalloproteinase Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Matrix Metalloproteinases/chemistry , Animals , Drug Delivery Systems , Humans , Matrix Metalloproteinases/metabolismABSTRACT
The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activity and we show that selected fragments inhibit MET downstream signalling. Thus we demonstrate that targeting the lysine-binding pocket of NK1 is an effective strategy to generate MET receptor antagonists and we offer proof of concept that the HGF/SF-MET interface may be successfully targeted with small molecules. These studies have broad implications for the development of HGF/SF-MET therapeutics and cancer treatment.
ABSTRACT
The inhibition of a subgroup of human serine/threonine protein phosphatases is responsible for the cytotoxicity of cantharidin and norcantharidin against tumor cells. It is shown that the anhydride rings of cantharidin and norcantharidin are hydrolyzed when bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules are reported. Norcantharidin shows a unique binding conformation with the catalytically active Mn2PP5c, while cantharidin is characterized by a double conformation in its binding mode to the protein. Different binding modes of norcantharidin are observed depending of whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures will provide the basis for the rational design of new cantharidin-based drugs.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cantharidin/pharmacology , Enzyme Inhibitors/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cantharidin/chemistry , Hydrophobic and Hydrophilic Interactions , Phosphoprotein Phosphatases/chemistry , Protein Conformation , Structure-Activity RelationshipABSTRACT
The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.
Subject(s)
Metalloendopeptidases/chemistry , Protein Structure, Tertiary , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Humans , Matrix Metalloproteinase 10 , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Sequence AlignmentABSTRACT
The water proton nuclear magnetic relaxation dispersions (NMRD) of hexaaquochromium(III) in water and in water-glycerol solutions were obtained at several temperatures and viscosities. The data were analyzed in terms of the available theories by taking into account the contributions from first sphere, second sphere, and outer sphere water molecules. A meaningful analysis was possible by taking advantage of the structural model obtained from (17)O relaxation data, which was recently made available in the literature (Bleuzen, A.; Foglia, F.; Furet, E.; Helm, L.; Merbach, A.; Weber, J. J. Am. Chem. Soc. 1996, 118, 12 777). Dynamic parameters, like the molecular rotational time, the exchange time of the water protons of the first coordination sphere, the correlation time for electron relaxation, and the magnetic field dependence of electron relaxation were obtained. The possible contribution to water proton relaxivity of second sphere water molecules for some other hexaaqua complexes is also discussed.
