Biomedical subjects

L Scapozza

Publications and source records attributed to L Scapozza.

29 records · Page 2Linked to original sources

PubMed1998

Density functional studies on herpes simplex virus type 1 thymidine kinase-substrate interactions: the role of Tyr-172 and Met-128 in thymine fixation.

The enzyme herpes simplex virus type 1 thymidine kinase (HSV1 TK) salvages thymidine into the DNA metabolism of the virus. In the active site, the thymine ring of the nucleoside binds in a pocket, formed by two residues, Tyr-172 and Met-128, in a sandwich-type orientation. To investigate the nature of the thymine-enzyme pocket interactions, we have carried out density functional theory calculations with gradient-corrected exchange-correlation functionals of models of the thymine-HSV1 TK adduct. Our calculations indicate that the role of Met-128 in the substrate fixation is purely steric and hydrophobic, while the substrate-Tyr-172 interaction is essentially electrostatic in nature. These findings are completely consistent with the available catalytic properties of mutants on the 128 position.

Adenosine Triphosphate↗

PubMed1997

The bioactive conformation of neuropeptide Y analogues at the human Y2-receptor.

Several attempts to investigate the bioactive conformation of neuropeptide Y have been made so far. As cyclic peptides are much more rigid than linear ones, we decided to synthesise cyclic analogues of the C-terminal dodekapeptide amide neuropeptide Y Ac-25-36. Cyclisation was performed by side chain lactamisation of ornithine or lysine and glutamic or aspartic acid. The affinity of the 19 peptides ranged from Ki 0.6 nM to greater than 10,000 nM. We found that the size, position, orientation, configuration. and the location of the cycle plays an important role for receptor recognition. Circular dichroic studies have been performed to characterise the secondary structure of each peptide. Receptor binding studies were carried out on human neuroblastoma cell lines SK-N-MC (Y1) and SMS-KAN (Y2), and on rabbit kidney membranes (Y2). The pharmacological and spectral data showed that the alpha-helix content was not the predominant factor for high Y2-receptor affinity. Instead, the location and the size of the hydrophobic lactam bridge, and the conserved C-terminal tetrapeptide (Arg-Glu-Arg-Tyr) seemed to be the main parameters. Using molecular dynamics, the structures of four cyclic peptides (i,i+4) have been investigated and compared with the previously published NMR structure of one of the cyclic peptide analogues. Significant differences have been found in the overall three-dimensional fold of the peptides. The distances between the N- and the C-terminus allow discrimination between peptides with high binding affinity and those with low binding affinity, because of the correlation that was found with the measured affinity. Thus, this study suggests that a turn-like structure and the orientation of the C-terminus towards the N-terminus play major roles for high affinity binding of cyclic dodecapeptides to the Y2-receptor. None of the cyclic segments exhibits significant affinity to the Y1-receptor. Thus, these results support the hypothesis of a discontinuous binding site of neuropeptide Y at the Y1-receptor.

Animals↗

PubMed1996

Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor.

In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100-fold increase in affinity compared to the C-terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS-KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH-SY5Y cells and cyclic [Lys28-Glu32] NPY Ac-25-36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D-NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.

Amino Acid Sequence↗

PubMed1996

Allosteric regulation in a family of enterobacterial aspartate transcarbamylases: intramolecular transmission of regulatory signals in chimeric enzymes.

Several enterobacterial aspartate transcarbamylases (ATCases) exhibit a [2(C3):3(r2)] quaternary structure analogous to that of the Escherichia coli enzyme. Despite their conserved quaternary structures, these enzymes present substantial differences in the co-operativity of substrate binding and in their allosteric regulation by nucleotide effectors. A comparison between different enzymatic species provides an opportunity to expand our understanding of the molecular basis of allostery in ATCase. Chimeric ATCases were constructed by exchanging subdomain regions involved in quaternary structural features, such as the r1-c4 regulatory-catalytic subunit interface analyzed in this study, in order to define the involvement of this interface in the several components of allosteric regulation. The r1-c4 interface was found to constitute an essential element for the recognition and the transmission of the ATP regulatory signal in the Serratia marcescens and the Proteus vulgaris ATCases, as it does in the E. coli ATCase. Besides, the specific amino acid composition of the C-terminal region of the regulatory chain and its interactions with the amino acid residues in the 240s loop of the catalytic chain (r1-c4 interactions) were found to modulate the amplitude of the enzyme's response to ATP. The C-terminal region of the regulatory chain did not appear to participate directly in the regulation of the three native ATCases by CTP. Even when CTP acts as an activator, as in the P. vulgaris and S. marcescens ATCases, its signal follows a route distinct from that of the general activator ATP. Synergistic inhibition by CTP and UTP was found to involve the transmission of a specific UTP signal. This signal appeared different in the various ATCases, involving the C-terminal region of the regulatory chain in the E. coli and S. marcescens ATCases but not in the P. vulgaris ATCase.

Adenosine Triphosphate↗

PubMed1996

Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

Amino Acid Sequence↗

PubMed1995

Structure-based analysis of inhibitor binding to Ht-d.

A theoretical study was performed on the structure of both the native and inhibited metalloproteinase Ht-d (E.C. 3.4.24.42) solved at 2.0 A resolution. The energy maps calculated by program GRID clearly showed the extended binding site of Ht-d and allowed localization and characterization of the pockets S1-S3 and S1'-S3'. The GRID energy contour maps point out the particular shape of the S1' pocket in agreement with experimental density maps and inhibited Ht-d structures. Based on the high degree of sequence homology of the Ht-d active site to that of mammalian metalloproteinases, the characterization of active site pockets was extended to neutrophil collagenase, fibroblast collagenase, stromelysin 1 and 2. Thirty residues of the Ht-d propeptide were modeled and optimized with reference to the Ht-d structure, giving insight to the mechanism of natural inhibition in metalloproteinase proenzymes. Kinetic measurements of Ht-d inhibition by a series of synthetic peptides show, in agreement with our Ht-d propeptide model, the crucial role of cysteine and adjacent residues in the specificity of Ht-d propeptide. This study suggests the structural link between Ht-d and mammalian metalloproteinases, contributing to the understanding of the mechanism of natural and synthetic inhibitor binding to metalloproteinases. Therefore, Ht-d is a good model system for the design of novel inhibitors against these enzymes with enhanced potency and specificity.

Journal Article↗

PubMed1995

Molecular dynamics and structure-based drug design for predicting non-natural nonapeptide binding to a class I MHC protein.

Starting from the known three-dimensional structure of the class I major histocompatibility complex-encoded HLA-B*2705 protein, three non-natural nonapeptides were designed to fit optimally the HLA-B*2705-binding groove. The optimization was performed using structure-based drug design methods and the fact that all the possible interactions of the secondary anchor residue (position 3) with its human leukocyte antigen-binding pocket (pocket D) in nature are not entirely utilized. 150 ps molecular-dynamics (MD) simulation in water was employed to study the stability of the bimolecular complexes with three non-natural peptides (P3 = homophenylalanine, beta-naphthylalanine, alpha-naphthylalanine) as well as with the two natural homologues (P3 = Gly, Leu). Various structural and dynamical properties (atomic fluctuations, solvent-accessible surface areas, peptide Calpha-atom positions) of the simulated bimolecular complexes were used to compare the three non-natural with the two natural ligands. Since the various molecular properties have been shown previously to be related to the binding affinity of nonapeptide ligands to the major histocompatibility complex (MHC) HLA-B*2705 protein, the MD data predict a rather higher stability of MHC-ligand complexes with the three non-natural peptides, suggesting that the unnatural peptides studied show an enhanced binding affinity to the HLA-B*2705 protein. These results are in agreement with the experimental values of a semi-quantitative in vitro assembly assay, performed on the five nonapeptides (P3 = Gly, Leu, homophenylalanine, beta-naphthylalanine, alpha-naphthylalanine), which shows their ability to stabilize the native conformation of the HLA-B*2705 heavy chain and also shows that the three non-natural ligands bind with higher affinity (0.5 micro M) to the MHC protein than the two natural homologues (40 micro M). Thus, this study demonstrates that structural information combined with rational design and molecular-dynamics simulations can illustrate and predict MHC binding and potential T-cell epitope properties as well as contribute to the design of new non-peptidic MHC inhibitors that may be useful for the selective immunotherapy of autoimmune diseases to which HLA alleles are directly associated.

Journal Article↗

PubMed1995

Rational design of nonnatural peptides as high-affinity ligands for the HLA-B*2705 human leukocyte antigen.

From the three-dimensional structure of the class I major histocompatibility complex (MHC) HLA-B*2705 protein, several nonnatural peptides were designed either to optimize the interactions of one peptide amino acid (position 3) with its HLA binding pocket (pocket D) or to simplify the T-cell receptor-binding part by substitution with organic spacers. The stability of each MHC-ligand complex was simulated by 150-ps molecular dynamics in a water environment and compared with that of the natural complexes. All peptides were synthesized and tested for binding to the class I MHC protein in an in vitro assembly assay. As predicted from the computed atomic fluctuations and buried surface areas of MHC-bound ligands, bulky hydrophobic side chains at position 3 enhance the binding of a nonameric peptide to the HLA-B27 protein. Furthermore, it was possible to simplify half of the peptide sequence (residues 4-8) by replacement with organic fragments without altering the affinity of the designed ligands for the class I MHC protein. This study constitutes an initial step toward the rational design of nonpeptide class I MHC ligands for use in the selective immunotherapy of autoimmune diseases associated with particular HLA alleles.

Amino Acid Sequence↗

PubMed1994

Molecular dynamics simulation of MHC-peptide complexes as a tool for predicting potential T cell epitopes.

The class I major histocompatibility complex-encoded HLA-B*2705 protein was simulated in complex with six different peptides exhibiting unexpected structure-activity relationships. Various structural and dynamical properties of the solvated protein-peptide complexes (atomic fluctuations, solvent-accessible surface areas, hydrogen bonding pattern) were found to be in qualitative agreement with the available binding data. Peptides that have been experimentally shown to bind to the protein remained tightly anchored to the MHC molecule, whereas nonbinders were significantly more weakly complexes to the protein and progressively dissociate from it at their N- and C-terminal ends. The molecular dynamics simulations emphasize the unexpectedly important role of secondary anchors (positions 1 and 3) in influencing the MHC-bound conformation of antigenic nonapeptides. Furthermore, it confirms that dominant anchor residues cannot solely account for peptide binding to a class I MHC molecule. The molecular dynamics method could be used as a complementary tool to T cell epitope predictions from the primary sequences of proteins of immunological interest. It is better suited to MHC proteins for which a crystal structure already exists. Furthermore, it may facilitate the engineering of T cell epitopes as well as the rational design of new MHC inhibitors designed to fit optimally the peptide binding cleft.

Amino Acid Sequence↗

PubMed1994

A new quantum chemical approach in QSAR-analysis. Parametrisation of conformational energies into molecular descriptors JMn (steric) and JSn (electronic).

Two new types of structure-related molecular descriptors, JMn and JSn, have been developed using conformational energies from quantum chemical calculations. For this purpose propipocaine (CAS 3670-68-6) was chosen as a model and 42 analogues were studied. The quantum chemical calculations were performed applying AM1 and PCILO approximation methods. Appropriate mathematical models were designed to calculate steric parameter log JM1 and electronic parameters JS1 to JS6. The values obtained for these parameters were used in multiple linear regression analysis for the evaluation of the structure-activity relationship. Furthermore, a comparison between electronic parameters JSn and sigma (Hammett) was made. The results show, that these parameters can be used successfully in predicting the biological activity of compounds in this model. Although, JS5 values are comparable to sigma-Hammett, the electronic parameter JS2 gives a better correlation in QSAR-analysis involving two parameters JS2 and log JM1.

Anesthetics, Local↗

PubMedDate not supplied

Integrated homology modelling and X-ray study of herpes simplex virus I thymidine kinase: a case study.

Knowledge-based homology modelling together with site-directed mutagenesis, epitope and conformational mapping is an approach to predict the structures of proteins and for the rational design of new drugs. In this study we present how this procedure has been applied to model the structure of herpes simplex virus type 1 thymidine kinase (HSV1 TK, HSV1 ATP-thymidine-5'-phosphotransferase, EC 2.7.1.21). We have used, and evaluated, several secondary structure prediction methods, such as the classical one based on Chou and Fastman algorithm, neural networks using the Kabsch and Sander classification, and the PRISM method. We have validated the algorithms by applying them to the porcine adenylate kinase (ADK), whose three-dimensional structure is known and that has been used for the alignment of the TKs as well. The resulting first model of HSV1-TK consisted of the first beta-strand connected to the phosphate binding loop and its subsequent alpha-helix, the fourth beta-strand connected to the conserved FDRH sequence and two alpha-helix with basic amino acids. The 3D structure was built using the X-ray structure of ADK as template and following the general procedure for homology modelling. We extended the model by means of COMPOSER, an automatic process for protein modelling. Site-directed mutagenesis was used to experimentally verify the predicted active-site model of HSV1-TK. The data measured in our lab and by others support the suggestion that the FDRH motif is part of the active site and plays an important role in the phosphorylation of substrates. The structure of HSV1 TK, recently solved in collaboration with Prof. G. Schulz at 2.7 A resolution, includes 284 of 343 residues of the N-terminal truncated TK. The secondary structures could be clearly assigned and fitted to the density. The comparison between crystallographically determined structure and the model shows that nearly 70% of the HSV1 TK structure has been correctly modelled by the described integrated approach to knowledge based ligand protein complex structure prediction. This indicate that computer assisted methods, combined with "manual" correction both for alignment and 3D construction are useful and can be successful.

Crystallography, X-Ray↗