Modulation of Wnt/ß-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer.

Using a screen for Wnt/ß-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe(2+) with a dissociation constant of ∼10(-19) M, with much weaker binding to Fe(3+) and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific ß-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on ß-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse ß-catenin-independent cell lines. HQBA is a promising specific intracellular Fe(2+) chelator with activity against spontaneous mouse mammary cancers.

Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase.

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants.

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.

Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

S-nitrosothiol repletion by an inhaled gas regulates pulmonary function.

NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O(2)-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O(2)/O(2)(-). This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O(2) or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.

Multivalency effects in protein--carbohydrate interaction: the binding of the Shiga-like toxin 1 binding subunit to multivalent C-linked glycopeptides.

A series of monovalent and bivalent glycopeptides displaying a C-linked analogue of the Pk trisaccharide, the in vivo ligand for the pentavalent Shiga-like toxin binding subunit (SLT-1B), were prepared and evaluated as ligands for SLT-1B by isothermal titration microcalorimetry and competitive enzyme-linked immunosorbent assay (ELISA). Although none of the monovalent ligands showed any enhancement in affinity compared to O-methyl glycoside, two bivalent ligands show significant enhancements in affinity in assays. This observation represents the first calorimetric observation of an enhancement in affinity for this system. In contrast, only one of the two ligands shows an enhancement in the competitive ELISA. Together, these data signal a difference in the means by which the two ligands achieve affinity, apparently triggered by a change in the nature of the linker domain. These results provide a rationalization for apparently contradictory reports from the recent literature and again emphasize the importance of investigating complex binding phenomena by multiple techniques.

Enzyme-catalyzed synthesis of carbohydrates.

Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.

Microcalorimetric determination of thermodynamic parameters for ionophore-siderophore host-guest complex formation.

Thermodynamic parameters (delta H, delta S, and delta G) were determined by microcalorimetry in wet chloroform for host-guest assembly formation involving second-sphere complexation of the siderophore ferrioxamine B by crown ether (18-crown-6, cis-dicyclohexano-18-crown-6, benzo-18-crown-6) and cryptand (2.2.2 cryptand) hosts. Similar data were also collected for the same hosts with the pentylammonium ion guest, which corresponds to the pendant pentylamine side chain of ferroxamine B. Host-guest assembly formation constants (Ka) obtained from microcalorimetry agree with values obtained indirectly from chloroform/water extraction studies in those cases where comparable data are available. On the basis of a trend established by the pentylammonium guest, an enhanced stability relative to the crown ethers is observed for the assembly composed of ferrioxamine B and 2.2.2 cryptand that is due to entropic effects. Trends in delta H and delta S with changes in host and guest structure are discussed and attributed directly to host-guest complex formation, as solvation effects were determined to be insignificant (delta Cp = 0).

Initiating a structural study of 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1. 2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was purified by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 A on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 A.

Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis.

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5, 293. 5 K. Crystals are orthorhombic, belonging to space group C2221 with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 A. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimer in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 A have been collected and the structure determined by the molecular replacement method.

Exploring the basis of peptide-carbohydrate crossreactivity: evidence for discrimination by peptides between closely related anti-carbohydrate antibodies.

To investigate the molecular basis of antigenic mimicry by peptides, we studied a panel of closely related mAbs directed against the cell-wall polysaccharide of group A Streptococcus. These antibodies have restricted V-gene usage, indicating a shared mechanism of binding to a single epitope. Epitope mapping studies using synthetic fragments of the cell-wall polysaccharide supported this conclusion. All of the mAbs isolated crossreactive peptides from a panel of phage-displayed libraries, and competition studies indicated that many of the peptides bind at or near the carbohydrate binding site. Surprisingly, the peptides isolated by each mAb fell into distinct consensus-sequence groups that discriminated between the mAbs, and in general, the peptides bound only to the mAbs used for their isolation. Similar results were obtained with polyclonal antibodies directed against synthetic oligosaccharide fragments of the streptococcal cell-wall polysaccharide. Thus, the peptides appear to be specific for their isolating antibodies and are not recognized by the same mechanism as their carbohydrate counterparts.

A comparison of the fine saccharide-binding specificity of Dioclea grandiflora lectin and concanavalin A.

The lectin from the seeds of Dioclea grandiflora (DGL) is a Man/Glc-specific tetrameric protein with physical and saccharide-binding properties reported to be similar to that of the jack bean lectin concanavalin A (ConA). Unlike other plant lectins, both DGL and ConA bind with high affinity to the core trimannoside moiety, 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, which is present in all asparagine-linked carbohydrates. In the present study, hemagglutination inhibition techniques have been used to investigate binding of DGL and ConA to a series of mono- and dideoxy analogs of methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and to a series of asparagine-linked oligomannose and complex oligosaccharides and glycopeptides. The results indicate that both DGL and ConA recognize epitopes on all three residues of the trimannoside: the 3-, 4-, and 6-hydroxyl groups of the alpha(1-6)Man residue, the 3-hydroxyl group of the alpha(1-3)Man residue, and the 2- and 4-hydroxyl groups of the central Man residue of the core trimannoside. However, unlike ConA, DGL does not bind to biantennary complex carbohydrates. This was confirmed by showing that biantennary complex glycopeptides do not bind to a DGL-Sepharose affinity column. Unlike ConA, DGL does not show enhanced affinity for a large N-linked oligomannose carbohydrate (Man9 glycopeptide) relative to the trimannoside. Thus, DGL and ConA share similar epitope recognition of the core trimannoside moiety. However, they exhibit differences in their fine specificities for larger N-linked oligomannose and complex carbohydrates.

Analysis of the binding specificities of oligomannoside-binding proteins using methylated monosaccharides.

The binding specificities of the closely related lectins from Canavalia ensiformis and Dioclea grandiflora were examined using specifically O-alkylated mono- and disaccharides. Both lectins accept any substitution at the monosaccharide C2 hydroxyl group. The binding energy of C2-alkylated ligands-concanavalin A complexes increases by 1 kcal mol-1 for the C2-O-ethyl ligand, while the binding energies of the corresponding complexes with the Dioclea lectin are identical. Both lectins accept methyl, but not ethyl, substitution of the C3 hydroxyl, in contrast to earlier reports. The results are interpreted in terms of existing models of the concanavalin A binding site. While the results are consistent with a model of the concanavalin A extended binding site that places the non-reducing terminus of all disaccharides in the monosaccharide binding site, they point to the dangers of interpreting the binding behavior of unnatural saccharide ligands on the basis of crystallographic data obtained with native ligands.

Specificity of C-glycoside complexation by mannose/glucose specific lectins.

The binding of the mannose/glucose specific lectins from Canavalia ensiformis (concanavalin A) and Dioclea grandiflora to a series of C-glucosides were studied by titration microcalorimetry and fluorescence anisotropy titration. These closely related lectins share a specificity for the trimannoside methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, and are a useful model system for addressing the feasibility of differentiating between lectins with overlapping carbohydrate specificities. The ligands were designed to address two issues: (1) how the recognition properties of non-hydrolyzable C-glycoside analogues compare with those of the corresponding O-glycosides and (2) the effect of presentation of more than one saccharide recognition epitope on both affinity and specificity. Both lectins bind the C-glycosides with affinities comparable to those of the O-glycoside analogues; however, the ability of both lectins to differentiate between gluco and manno diastereomers was diminished in the C-glycoside series. Bivalent norbornyl C-glycoside esters were bound by the lectin from Canavalia but only weakly by the lectin from Dioclea. In addition to binding the bivalent ligands, concanavalin A discriminated between C-2 epimers, with the manno configuration binding more tightly than the gluco. The stoichiometry of binding of the bivalent ligands to both di- and tetrameric lectin was two binding sites per ligand, rather than the expected 1:1 stoichiometry. Together, these results suggest that concanavalin A may possess more than one class of carbohydrate binding sites and that these additional sites show stereochemical discrimination similar to that of the previously identified monosaccharide binding site. The implications of these findings for possible in vivo roles of plant lectins and for the use of concanavalin A as a research tool are discussed.

Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.

Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities.

The thermodynamics of binding of a system of plant lectins specific for the oligosaccharide methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside have been studied calorimetrically. This system of lectins consists of concanavalin A, the lectin isolated from Dioclea grandiflora, and the lectin from Galanthus nivalis. The group thus contains lectins with similar structures and similar binding properties as well as lectins with different structures but similar binding properties. Concanavalin A and the lectin from Dioclea are highly homologous, while the lectin from Galanthus nivalis shares no sequence homology with either of the legume lectins, although it also binds the mannose trisaccharide tightly. Calorimetric data for oligosaccharide binding to both of the legume lectins suggests that the total binding site comprises a single high-affinity site and an additional extended site. The pattern of binding for the lectin from Galanthus is significantly different. Binding studies with the same saccharides indicate that the lectin has binding sites designed specifically for the 1-->3 and 1-->6 arms of the mannose trisaccharide that are unable to accommodate other saccharides. Enthalpy--entropy compensation was observed for several saccharides as a function of lectin structure. Contributions of solvation effects to the enthalpy of binding and the configurational entropies were determined experimentally. For those systems studied here, solute-solute attractive interactions and configurational entropies were the greatest contributors to enthalpy-entropy compensation. Our studies clearly demonstrate that, despite their common affinity for the mannose trisaccharide, the three lectins bind oligosaccharides very differently.

Interaction of the Shiga-like toxin type 1 B-subunit with its carbohydrate receptor.

A study of the binding of the Shiga-like toxin 1 (SLT-1) to the P(k) trisaccharide [methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D- glucopyranoside] and its constituent dissacharides was carried out. The trisaccharide represents the carbohydrate recognition domain of the neutral glycolipid receptor of the SLT-1, globotriosylceramide (GbOse3). The binding constant for soluble trisaccharide to the soluble pentameric B-subunit is weak, with a K(a) of (0.5-1) x 10(3) M-1 for B-subunit monomer. Scatchard analysis of the binding data indicates five identical non-interacting carbohydrate binding sites per B-subunit pentamer and no cooperativity in binding. Despite weak binding (delta G = -3.6 kcal mol-1), the enthalpy of binding (delta H = -12 kcal mol-1) and the change in molar heat capacity accompanying binding (delta C(p) = -40 eu) are comparable to other protein-carbohydrate interactions. Dynamic light scattering studies indicate that carbohydrate binding induces protein aggregation. At carbohydrate concentrations where > 90% of B-subunit monomers are bound, the far-UV CD spectra were unchanged, whereas a change in the near-UV CD, maximal near 270 nm, titrated to give an apparent binding constant in good agreement with that obtained by isothermal microcalorimetry. Steady-state fluorescence and fluorescence lifetime measurements indicated that the environments of the central tryptophans are perturbed during saccharide binding, and the changes correlate with the extent of protein aggregation. On the basis of the thermodynamics of binding, optical spectroscopy, and binding-induced aggregation, we propose a model of SLT-1-membrane interaction that relies on protein-carbohydrate interaction for specificity and protein-lipid interaction for tight binding.

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation.

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.