Sialic acid C-glycosides with aromatic residues: investigating enzyme binding and inhibition of Trypanosoma cruzi trans-sialidase

Several α-configured C-sialosides were synthesised by cross metathesis and further synthetic derivatisation to obtain ligands for Trypanosoma cruzi trans-sialidase (TcTS), a key enzyme in Chagas disease. Affinities of these compounds to immobilised TcTS were measured by surface plasmon resonance (SPR). The K(D) values thus obtained are in the lower millimolar range and will be discussed. The results show the importance of addressing Tyr(119) and Trp(312) side chains of TcTS in target oriented ligand synthesis, since these amino acids constitute the acceptor binding region in the active site of TcTS. The best ligand showed a significant decrease of TcTS activity in a preliminary NMR based inhibition assay.     

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.     

Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I

Background: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyp hosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides.Results: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P₂). The P-1-tethered D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of Ptdlns(4,5)P₂ but not with Ins(1,4,5)P₃. The acyl-modified Ptdlns(4,5)P₂ analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P₂ phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [³H]BZDC-Ins(1,4,5)P₃-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix.Conclusions: The Ptdlns(4,5)P₂-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of Ptdlns(4,5)P₂ and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.     

Probing mode and site of substrate water binding to the oxygen-evolving complex in the S2 state of photosystem II by 17O-HYSCORE spectroscopy

In the oxygen-evolving complex (OEC) of photosystem II (PSII) molecular oxygen is formed from two substrate water molecules that are ligated to a mu-oxo bridged cluster containing four Mn ions and one Ca ion (Mn4OxCa cluster; Ox symbolizes the unknown number of mu-oxo bridges; x >or= 5). There is a long-standing enigma as to when, where, and how the two substrate water molecules bind to the Mn4OxCa cluster during the cyclic water-splitting reaction, which involves five distinct redox intermediates (Si-states; i = 0,...,4). To address this question we employed hyperfine sublevel correlation (HYSCORE) spectroscopy on H217O-enriched PSII samples poised in the paramagnetic S2 state. This approach allowed us to resolve the magnetic interaction between one solvent exchangeable 17O that is directly ligated to one or more Mn ions of the Mn4OxCa cluster in the S2 state of PSII. Direct coordination of 17O to Mn is supported by the strong (A approximately 10 MHz) hyperfine coupling. Because these are properties expected from a substrate water molecule, this spectroscopic signature holds the potential for gaining long-sought information about the binding mode and site of one of the two substrate water molecules in the S2 state of PSII.     

Identification of photolabeled peptides for the acceptor substrate binding domain of beta 1,4-galactosyltransferase.

We successfully applied a carbene-generating N-acetylglucosamine derivative carrying a biotinyl group to the radioisotope-free identification of peptides within bovine UDP-galactose: N-acetylglucosamine beta 1,4-galactosyltransferase (GalT, EC 2.4.1.38) catalytic domain. Owing to the low yield of cross-linking, conventional photoaffinity labeling experiments usually encounter a thorny problem in attempting to isolate labeled components from very complex mixtures. A biotin tag introduced with our photoaffinity probe enabled us to separate the photolabeled protein from a large amount of coexisting unlabeled GalT. The introduction of biotin was also useful for the radioisotope-free detection of a labeled protein based on a highly sensitive chemiluminescent technique. We developed a novel poly(vinylidene difluoride) membrane for the identification of labeled peptides in a simple dot blot assay. Using this membrane, we successfully identified biotinyl peptides among a number of HPLC separated fragments derived from the protease digestion of photolabeled GalT proteins. The sequence analysis revealed that the biotin tag was incorporated within a tryptic GalT fragment of Y197-R208. Our approach yields, for the first time, information on the acceptor substrate binding-site fragment in this enzyme, that has been difficult to obtain using other approaches. These data are consistent with previous suggestions concerning the GalT acceptor site and clearly demonstrate the effectiveness of our approach for rapid identification of photolabeled peptides.     

Dynamic combinatorial carbohydrate libraries: probing the binding site of the concanavalin A lectin

Dynamic combinatorial chemistry (DCC) has emerged as an efficient approach to receptor/ligand identification based on the generation of combinatorial libraries by reversible interconversion of the library constituents. In this study, the implementation of such libraries on carbohydrate-lectin interactions was examined with the plant lectin Concanavalin A as a target species. Dynamic carbohydrate libraries were generated from a pool of carbohydrate aldehydes and hydrazide linker/scaffold components through reversible acylhydrazone exchange, resulting in libraries containing up to 474 constituents. Dynamic deconvolution allowed the efficient identification of the structural features required for binding to Concanavalin A and the selection of a strong binder, a tritopic mannoside, showing an IC(50)-value of 22 microM.     

Probing the dynamic nature of water molecules and their influences on ligand binding in a model binding site

The model binding site of the cytochrome c peroxidase (CCP) W191G mutant is used to investigate the structural and dynamic properties of the water network at the buried cavity using computational methods supported by crystallographic analysis. In particular, the differences of the hydration pattern between the uncomplexed state and various complexed forms are analyzed as well as the differences between five complexes of CCP W191G with structurally closely related ligands. The ability of docking programs to correctly handle the water molecules in these systems is studied in detail. It is found that fully automated prediction of water replacement or retention upon docking works well if some additional preselection is carried out but not necessarily if the entire water network in the cavity is used as input. On the other hand, molecular interaction fields for water calculated from static crystal structures and hydration density maps obtained from molecular dynamics simulations agree very well with crystallographically observed water positions. For one complex, the docking and MD results sensitively depend on the quality of the starting structure, and agreement is obtained only after redetermination of the crystal structure and refinement at higher resolution.     

Theoretical studies on substrate binding to the active site of carbonic anhydrase

The role of some amino acids and metal ions in the catalytic activity of carbonic anhydrase (carbonate dehydratase EC 4.2.1.1) has been investigated. The additional stabilization of the transition state complex was used as the qualitative measure of the effect of molecular surrounding on CO₂ hydration reaction calculated within the approximate CNDO /2 approach. The effect of the molecular environment has been simulated by inclusion into the SCF LCAO MO Hamiltonian, a term representing the interaction with a set of point charges and atomic dipoles centered on experimentally determined position of Zn²⁺ ion and all atoms of histidine 94, 96, 119 and threonine 199, 200 (or histidine 200 in the case of carbonic anhydrase B). The possible molecular mechanism of CO₂ hydration inside the active site has been also discussed.     

Analysis of the importance of the metallo-beta-lactamase active site loop in substrate binding and catalysis

The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and molecular modeling. The W64A mutation was performed in IMP-1 and BcII (after replacement of the BcII 60-66 peptide by that of IMP-1) and always resulted in increased K(i) and K(m) and decreased k(cat)/K(m) values, an effect reinforced by complete deletion of the loop. k(cat) values were, by contrast, much more diversely affected, indicating that the loop does not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand is also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications.     

Building addressable libraries: site selective coumarin synthesis and the "real-time" signaling of antibody-coumarin binding

[reaction: see text] The feasibility of using active semiconductor chips containing addressable arrays of microelectrodes for the "real-time" monitoring of biologically relevant binding events has been demonstrated by detecting the binding of a coumarin substrate by an anticoumarin antibody. The coumarin substrate was synthesized proximal to predetermined electrodes on the chip with the use of a Pd(II) reagent that was itself generated by using the selected electrodes. Once the coumarin was synthesized, its binding to the anticoumarin antibody was detected by monitoring the current associated with a ferrocene-ferrocinium ion redox cycle that was established between the electrodes on the chip and a remote auxiliary electrode.     

Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome

The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.     

The mechanism of nitrogenase. Computed details of the site and geometry of binding of alkyne and alkene substrates and intermediates

The chemical mechanism by which the enzyme nitrogenase effects the remarkable reduction of N(2) to NH(3) under ambient conditions continues to be enigmatic, because no intermediate has been observed directly. Recent experimental investigation of the enzymatic consequences of the valine --> alanine modification of residue alpha-70 of the component MoFe protein on the reduction of alkynes, together with EPR and ENDOR spectroscopic characterization of a trappable intermediate in the reduction of propargyl alcohol or propargyl amine (HCC[triple bond]C-CH(2)OH/NH(2)), has localized the site of binding and reduction of these substrates on the FeMo-cofactor and led to proposed eta(2)-Fe coordination geometry. Here these experimental data are modeled using density functional calculations of the allyl alcohol/amine intermediates and the propargyl alcohol/amine reactants coordinated to the FeMo-cofactor, together with force-field calculations of the interactions of these models with the surrounding MoFe protein. The results support and elaborate the earlier proposals, with the most probable binding site and geometry being eta(2)-coordination at Fe6 of the FeMo-cofactor (crystal structure in the Protein Database), in a position that is intermediate between the exo and endo coordination extremes at Fe6. The models described account for (1) the steric influence of the alpha-70 residue, (2) the crucial hydrogen bonding with Nepsilon of alpha-195(His), (3) the spectroscopic symmetry of the allyl-alcohol intermediate, and (4) the preferential stabilization of the allyl alcohol/amine relative to propargyl alcohol/amine. Alternative binding sites and geometries for ethyne and ethene, relevant to the wild-type protein, are described. This model defines the location and scene for detailed investigation of the mechanism of nitrogenase.     

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsor-alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi , the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm². We note that while the infectivity of the parasite is eliminated at 4.2 J/cm^Z the trypomastigote motility continues for at least 16 h post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of ³H-AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi , the causative agent of Chagas disease.     

Hydrogen-bonded assembly and binding affinity of the multi-point acceptor and isophthalic acid

Supermolecular complexes formed by oligophenyleneethynylene derivatives and isophthalic acid were studied using AM1 method to obtain binding energy. Electronic spectra and IR spectra of the complexes were calculated by INDO/CIS and AM1 methods based on AM1 geometries. Results indicated that the dimer could be formed by the monomers via hydrogen bonding because of the negative binding energy. Binding energy of the complexes was affected by electronegativity and steric effects of the substituents. The first UV absorptions and IR frequencies of N-H bonds of the complexes were both red-shifted compared with those of the monomers. The complexes could bind small molecules via hydrogen bonds, resulting in the change in UV absorptions and an increase in IR frequencies of N-H bonds.     

Heteroleptic Oxidovanadium(V) Complexes with Activity against Infective and Non-Infective Stages of Trypanosoma cruzi

Five heteroleptic compounds, [V^VO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC₅₀ values of 0.29–3.02 μM. IN ligand and the new [V^VO₂(IN-H)] complex showed negligible activity. The most active compound [V^VO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [V^VO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC₅₀ values of [V^VO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 μM concentration of [V^VO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.     

The electron acceptor and catalytic properties of zirconium dioxide modified with aluminum and gallium oxides

The electron acceptor properties of the mixed oxide systems 20 mol % M₂O₃-ZrO₂ (M = Al, Ga) and 10 mol % M₂O₃-10 mol % Al₂O₃-ZrO₂ (M = Ga, Y) were studied by recording the EPR spectra of paramagnetic complexes of adsorbed anthraquinone. The EPR data were compared with the catalytic activity of the mixed oxides in the decomposition of propanols and butanols in a pulsed microcatalytic reactor. The Al₂O₃-ZrO₂ and Ga₂O₃-ZrO₂ solid solutions were found to be metastable and decompose as the temperature of calcining increased or a third component was introduced. Correspondence between the EPR data and catalytic experiment results was established. This correspondence substantiates the occurrence of the segregation of the Al₂O₃ and Ga₂O₃ amorphous phases on the surface of zirconia, which accompanies the decay of the M₂O₃-ZrO₂ (M = Al, Ga) metastable solid solutions. The Al₂O₃-ZrO₂ solid solution decomposes more readily than Ga₂O₃-ZrO₂.     

Using peptidic inhibitors to systematically probe the S1' site of caspase-3 and caspase-7

Fifteen ketone-containing peptides were designed, synthesized, and used to probe the effect of substitution at the P1' position on caspase-3 and -7 inhibition. Even with the large bias of Ac-Asp-Glu-Val-Asp at the P4-P1 positions, certain peptides with cyclic functionality in the P1' position show a dramatically reduced ability to inhibit these caspases. Additionally, trends toward isozyme selectivity were also uncovered for particular P1' substituents. The data indicate that substitution in the P1' position can drastically affect both caspase inhibition and selectivity. [structure: see text]     

Pyrene.pyrene complexes at the active site of cytochrome P450 3A4: evidence for a multiple substrate binding site

Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.     

A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.