A systematic method for analysing the protein hydration structure of T4 lysozyme.

A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water-protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters.

Peptide inhibitors of HIV-1 and HIV-2 proteases: a comparative study.

HIV-1 and HIV-2 proteases (PR) which play the key role in the formation of infectious viral particles offer a target for inhibitors that could block the maturation step. Inhibitors o HIV-1 PR exhibit mostly 1-2 orders of magnitude weaker affinity for HIV-2 PR. The subsite specificity study of the HIV-1 and HIV-2 proteases performed with inhibitors varying in the type of nonhydrolysable bonds and amino acid residues in the P1, P1'and P2'positions has led us to the design of inhibitors with 2S,4S and 2R,4S stereomeres of the hydroxyethylene isostere and Glu or Gln in the P2'positions. These compounds inhibit HIV-1 and HIV-2 proteases in vitro in subnanomolar concentrations and exhibit the activity in tissue culture.

An empirical potential study of the interaction of L-lysine-L-alanine-L-alanine tripeptide with four models of B-DNA with different compositions.

The empirical potential including the intra- and intermolecular energy terms was used to study the interaction of L-Lysine-L-Alanine-L-Alanine Tripeptide with four models of B-DNA with different compositions. On the basis of a detailed search of the respective potential energy surface, it was found that the peptide is preferentially bounded to the AT-rich sequences. Analysis of the different energy contributions indicated that the electrostatic term is responsible for this preference. The results agree with the experimental data on the selectivity of some DNA--binding proteins and polypeptides to AT-rich DNA.

Molecular dynamics study of the effect of the gamma-Abu insert on the conformational behavior of the glycopeptide dendrimers based on the oligolysine scaffold in N, N'-dimethylformamide.

Glycodendrimers bearing Tn (alpha-D-GalNAc-(1 --> O)-Ser/Thr), an identified tumor-associated carbohydrate antigen, hold promise in the post-surgery treatment of a variety of tumors such as metastatic breast cancer. We used molecular dynamics (MD) techniques to examine structural differences taking place during synthesis of two classes of tetravalent Multiple Antigen Glycopeptides (MAG) that differ only by the gamma-Abu insert in the structure of the oligolysine core. Each of the selected intermediates of the synthesis was modeled, subjected to the 2 ns run in N,N'-dimethylformamide (DMF) and geometrically characterized. We characterized: a) distances of free, or extended termini from the anchor, b) interatomic distances between free or substituted N termini, c) radius of gyration and d) spatial distribution of molecular density. A detailed conformational analysis of 16 glycodendrimers shows the distinct behavior of the inserted vs. non-inserted constructs already during the first steps of the modeled synthesis. It suggests that the character as well as the length of the insert has a major impact on the spatial characteristics and behavior of the dendritic molecules. The inserts can, in principle, increase a tendency of dendrimers to establish a high-density core, which is similar to the effect of a higher generation.

Coordination geometries of selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+) in metalloproteins.

In order to determine preferred coordination geometries of six divalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+), two sources of experimental data were exploited: Protein Data Bank and Cambridge Structural Database. Metal-binding sites of approximately 100 metalloproteins and 3000 smaller transition metal complexes were analyzed and classified. The correlation between the geometries of small-molecule crystal structures and the metal-binding sites in metalloproteins was investigated. The abundance of amino acid residues participating in coordination metal-protein bonds of metalloproteins was evaluated. From the performed analysis it follows that the octahedral arrangement is preferred by Co2+ and Ni2+, tetrahedral by Zn2+, square planar by Cu2+, and linear by Hg2+. Cadmium (II) cation tends to bind in both tetrahedral and octahedral arrangements and single coordination geometry cannot be unambiguously ascribed to it.

Structure and IR spectrum of phenylalanyl-glycyl-glycine tripetide in the gas-phase: IR/UV experiments, ab initio quantum chemical calculations, and molecular dynamic simulations.

We investigated the potential-energy surface (PES) of the phenylalanyl-glycyl-glycine tripeptide in the gas phase by means of IR/UV double-resonance spectroscopy, and quantum chemical and statistical thermodynamic calculations. Experimentally, we observed four conformational structures and we recorded their IR spectra in the spectral region of 3000-4000 cm(-1). Computationally, we investigated the PES by a combination of molecular dynamics/quenching procedures with high-level correlated ab initio calculations. We found that neither empirical potentials nor various DFT functionals provide satisfactory results. On the other hand, the approximative DFT method covering the dispersion energy yields a reliable set of the most stable structures, which we subsequently investigated with an accurate, correlated ab initio treatment. The global minimum contains three moderately strong intramolecular hydrogen bonds and is mainly stabilized by London dispersion forces between the phenyl ring, the carboxylic acid group, and various peptide bonds. A proper description of the last type of interaction requires accurate correlated ab initio calculations, including the complete basis set limit of the MP2 method and CCSD(T) correction terms. Since in our beam experiments the conformations are frozen by cooling from a higher temperature, it is necessary to localize the most stable structures on the free-energy surface rather than on the PES. We used two different procedures (rigid rotor/harmonic oscillator/ideal gas approximation based on ab initio characteristics and evaluation of relative populations from the molecular dynamic simulations using the AMBER potential) and both yield four structures, the global minimum and three local minima. These four structures were among the 15 most energetically stable structures obtained from accurate ab initio optimization. The calculated IR spectra for these four structures agree well with the experimental frequencies, which validates the localization procedure.

Inhibitors of HIV-1 protease: a major success of structure-assisted drug design.

Retroviral protease (PR) from the human immunodeficiency virus type 1 (HIV-1) was identified over a decade ago as a potential target for structure-based drug design. This effort was very successful. Four drugs are already approved, and others are undergoing clinical trials. The techniques utilized in this remarkable example of structure-assisted drug design included crystallography, NMR, computational studies, and advanced chemical synthesis. The development of these drugs is discussed in detail. Other approaches to designing HIV-1 PR inhibitors, based on the concepts of symmetry and on the replacement of a water molecule that had been found tetrahedrally coordinated between the enzyme and the inhibitors, are also discussed. The emergence of drug-induced mutations of HIV-1 PR leads to rapid loss of potency of the existing drugs and to the need to continue the development process. The structural basis of drug resistance and the ways of overcoming this phenomenon are mentioned.

Magnesium and biological activity of oxytocin analogues modified on aromatic ring of amino acid in position 2.

For the purpose of evaluating substitution effects in the ortho, meta or para positions of the aromatic ring of tyrosine or phenylalanine in position 2 of oxytocin on uterotonic activity in vitro in the presence and absence of magnesium ions, six new analogues of oxytocin ([D- and L-m-methylphenylalanine2]oxytocin, [D- and L-m-methoxyphenylalanine2]oxytocin and [D- and L-o-methyltyrosine2]-oxytocin) were synthesized and several previously described analogues resynthesized. For the phenylalanine series, it is found that, in the absence of magnesium ions, substitution of the ortho and meta positions leads to loss of intrinsic activity (the analogues are antagonists) in contrast to the para position. In the tyrosine series, only methyl substitution in the meta position has this effect (substitution of ortho position only attenuates the agonistic biological activity). Addition of Mg ions restores to a certain degree the agonistic activity in the case of the o-methylphenylalanine analogue and enhances the agonistic activity of o-methyltyrosine oxytocin. All other analogues keep the original qualities as in the absence of Mg. Molecular modelling calculations of the structure of the above analogues was carried out to help explain these findings of the molecular level.

A modular approach to HIV-1 proteinase inhibitor design.

HIV-1 proteinase represents a promising target for antiviral chemotherapy. We have designed, synthesized, and tested modular inhibitors combining an active-site inhibitor tethered to a structure targeted to the dimerization domain of the enzyme. At pH 5 the parent active site inhibitor, the equimolar mixture of active site and dimerization inhibitors, and the best compound from our series of modular inhibitors show the same inhibition activity. At neutral pH, however, the combination of the dimerization and active-site inhibitors shows a synergistic effect. Moreover, the modular inhibitor has an IC50 value 5x lower than the parent active site inhibitor and 2x lower than the equimolar mixture of the two parent inhibitors. The Lineweaver-Burk plot for modular inhibitors corresponds to a pattern for mixed type inhibition.

A picomolar inhibitor of resistant strains of human immunodeficiency virus protease identified by a combinatorial approach.

In order to identify inhibitors of various drug-resistant forms of the human immunodeficiency virus protease (HIV PR), we have designed and synthesized pseudopeptide libraries with a general structure Z-mimetic-Aa1-Aa2-NH2. Five different chemistries for peptide bond replacement have been employed and the resulting five individual sublibraries tested with the HIV PR and its drug-resistant mutants. Each mutant contains amino acid substitutions that have previously been shown to be associated with resistance to protease inhibitors, including Ritonavir, Indinavir, and Saquinavir. We have mapped the subsite preferences of resistant HIV PR species with the aim of selecting a pluripotent pharmaceutical lead. All of the enzyme species in this study manifest clear preference for an L-Glu residue in the P2' position. Slight, but significant, differences in P3' subsite specificity among individual resistant PR species have been documented. We have identified three compounds, combining the most favorable features of the inhibitor array, that exhibit low-nanomolar or picomolar Ki values for all three mutant PR species tested.

Potency comparison of peptidomimetic inhibitors against HIV-1 and HIV-2 proteinases: design of equipotent lead compounds.

HIV-1 and HIV-2 proteinases (PR) are responsible for the processing of viral polyproteins, a step that is crucial for the formation of infectious virus particles. PR represents one of the most important targets for antiviral chemotherapy. Inhibitors of HIV-1 PR usually exhibit a 10- to 100-fold weaker affinity for HIV-2 PR. In order to design subnanomolar inhibitors for both HIV-1 and HIV-2 PRs, we prepared a series of compounds varying in the type of scissile bond replacement as well as in the P1, P1', and P2' side chains. While inhibitors containing reduced amide, hydroxyethylamine and statine isosteres had Ki values in the range of 10(-10)-10(-9) M against HIV-1 PR; their activities against HIV-2 PR were several orders of magnitude lower. Glutamic acid was identified to be the optimal P2' residue for both PRs. HIV-2 PR was shown to be more sensitive to P2' Glu-->Gln replacement. Using this data set we were able to design and prepare hydroxyethylene isostere containing inhibitors that were equipotent against both PRs.

Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.

Configurations of diastereomeric hydroxyethylene isosteres strongly affect biological activities of a series of specific inhibitors of human-immunodeficiency-virus proteinase.

Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.