Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin

The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.     

Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.     

Large cyclic peptides as cores of multivalent ligands: application to inhibitors of receptor binding by cholera toxin

Large cyclic decapeptides (up to 50-atom ring) were synthesized efficiently on the solid phase with allyl-ester protection of the carboxyl terminus during elongation. Pentavalent ligands, in a "core-linker-finger" modular setup, were assembled by using these cyclic peptide cores to demonstrate large affinity gains for inhibition of surface receptor binding by the cholera toxin B pentamer. The results suggest that the peptide cores retain expanded conformation in solution so that shorter flexible linkers are needed for larger peptide cores to achieve the best inhibitory results.     

Synthesis and Langmuir studies of bivalent and monovalent alpha-D-mannopyranosides with lectin Con A

Highly avid interaction between carbohydrate ligands and lectin receptors nominally requires the ligand presentation in a clustered form. We present herein an approach involving Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface. Bivalent alpha-D-mannopyranoside containing the glycolipid ligand was used to study its binding profiles with lectin Con A, in comparison to the corresponding monovalent glycolipid. In addition to the bivalent and monovalent nature of the glycolipid ligands at the molecular level, the ligand densities at the monolayer level were varied with the aid of a nonsugar lipid molecule so as to obtain mixed monolayers with various sugar-nonsugar ratios. Lectin binding of bivalent and monovalent ligands at different ratios was monitored by differential changes in the surface area per molecule of the mixed monolayer, with and without the lectin. The present study shows that maximal binding of the lectin to the bivalent ligand occurs at lower sugar densities at the interface ( approximately 10% sugar in the mixed monolayer) than for that of the monovalent ligand ( approximately 20% sugar in the mixed monolayer). It is observed that complete coverage of the monolayer with only the sugar ligands does not allow all of the sugars to be functionally active.     

Optimization of tether length in nonglycosidically linked bivalent ligands that target sites 2 and 1 of a Shiga-like toxin

A series of bivalent ligands for a Shiga-like toxin have been synthesized, their experimentally determined inhibitory activities were compared with a simplified thermodynamic model, and computer simulations were used to predict the optimal tether length in bivalent ligands. The design of the inhibitors exploits the proximity of the C-2' hydroxyl groups of two P(k)-trisaccharides when bound to two different, neighboring carbohydrate recognizing binding sites located on the surface of Shiga-like toxin. NMR studies of the complex between the toxin and bivalent ligands show that site 2 and site 1 of a single B subunit are simultaneously occupied by a tethered P(k)-trisaccharide dimer. A simplified thermodynamic treatment provides the intrinsic affinities and binding energies for the intermolecular and intramolecular association events and permits the deconvolution of the contributions to the relative binding energies for the set of bivalent ligands. Conformational analysis based on MD simulations for bivalent galabioside dimers containing different tethers demonstrated that the calculated local concentrations of the pendant ligand at the second binding site correlate with the experimentally determined relative affinity values of the respective bivalent ligands, thereby providing a predictive method to optimize tether length.     

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.     

Multivalence and spot heterogeneity in microarray-based measurement of binding constants

Microarray technology is increasingly used for a miniaturised and parallel measurement of binding constants. In microarray experiments heterogeneous functionalization of surfaces with capture molecules is a problem commonly encountered. For multivalent ligands, especially, however, binding is strongly affected by receptor density. Here we show that high-resolution imaging of microarrays followed by image segmentation and separate analysis of bright and dark parts provides valuable information about ligand binding. Binding titrations were conducted with monovalent and bivalent fluorescent ligand peptides for the model receptor vancomycin. Microarrays were scanned with a confocal microscope and inhomogeneous spots were evaluated either as a whole or after segmentation into bright and dark areas. Whereas the binding constant for the monovalent ligand was hardly affected by spot heterogeneity, for the bivalent ligand affinity was higher for the parts of the spots with a greater density of receptors. This information was lost if the spots were analysed as a whole. These results reveal that imaging resolution may be a key factor in miniaturised binding assays, emphasising the importance of high-resolution images and image segmentation for new techniques, for example SPR imaging.     

Towards a rational spacer design for bivalent inhibition of estrogen receptor

Estrogen receptors are known drug targets that have been linked to several kinds of cancer. The structure of the estrogen receptor ligand binding domain is available and reveals a homodimeric layout. In order to improve the binding affinity of known estrogen receptor inhibitors, bivalent compounds have been developed that consist of two individual ligands linked by flexible tethers serving as spacers. So far, binding affinities of the bivalent compounds do not surpass their monovalent counterparts. In this article, we focus our attention on the molecular spacers that are used to connect the individual ligands to form bivalent compounds, and describe their thermodynamic contribution during the ligand binding process. We use computational methods to predict structural and entropic parameters of different spacer structures. We find that flexible spacers introduce a number of effects that may interfere with ligand binding and possibly can be connected to the low binding affinities that have been reported in binding assays. Based on these findings, we try to provide guidelines for the design of novel molecular spacers.     

Anchor-based design of improved cholera toxin and E. coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes

The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.     

Dependence of avidity on linker length for a bivalent ligand-bivalent receptor model system

This paper describes a synthetic dimer of carbonic anhydrase, and a series of bivalent sulfonamide ligands with different lengths (25 to 69 ? between the ends of the fully extended ligands), as a model system to use in examining the binding of bivalent antibodies to antigens. Assays based on analytical ultracentrifugation and fluorescence binding indicate that this system forms cyclic, noncovalent complexes with a stoichiometry of one bivalent ligand to one dimer. This dimer binds the series of bivalent ligands with low picomolar avidities (K(d)(avidity) = 3-40 pM). A structurally analogous monovalent ligand binds to one active site of the dimer with K(d)(mono) = 16 nM. The bivalent association is thus significantly stronger (K(d)(mono)/K(d)(avidity) ranging from ~500 to 5000 unitless) than the monovalent association. We infer from these results, and by comparison of these results to previous studies, that bivalency in antibodies can lead to associations much tighter than monovalent associations (although the observed bivalent association is much weaker than predicted from the simplest level of theory: predicted K(d)(avidity) of ~0.002 pM and K(d)(mono)/K(d)(avidity) ~ 8 × 10(6) unitless).     

Investigation of monovalent and bivalent enantioselective molecular recognition by electrospray ionization-mass spectrometry and tandem mass spectrometry

In this work is described the investigation of bivalent versus monovalent enantioselective molecular recognition in the context of enantioselective separations. Electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) are used for evaluating enantioselective systems through the measurement of (1) relative solution-phase binding constants via titration and (2) relative gas-phase binding via collision threshold dissociation. In HPLC, a cinchonane-type chiral stationary phase (CSP) based on tert.-butylcarbamoylquinine provides vastly increased retention and enantioselectivity for separation of bivalent versus monovalent alkoxy-benzoyl-N-blocked leucine enantiomers. The bivalent enantiomers are able to span and simultaneously interact with multiple interaction sites on the CSP surface, leading to enhanced separation. ESI-MS titration measurements also show an increased avidity for binding between bivalent selector and bivalent selectand, compared with the monovalent system. However, enhanced enantioselectivities measured in HPLC for the bivalent system cannot be reproduced by MS due to inherent mechanistic differences. Assumed discrepancies in relative response factors also give rise to systematic errors which are discussed. The results of MS/MS gas-phase experiments show that enantioselectivity is essentially lost in the absence of solvation, but that dissociation thresholds can provide a measure of relative dissociation energy in the bivalent interaction system compared to the monovalent counterpart. Such measurements may prove useful and efficient in better understanding multivalent interactions, in line with current theoretical considerations of effective concentrations and ion trap effects. This is the first application of mass spectrometric methods for assessing increased avidity of binding in multivalent enantioselective molecular recognition.     

Architectural and structural optimization of the protective polymer layer for enhanced targeting

Using Monte Carlo simulations we study the influence of ligand architecture (valence, branching length) and structure (polydispersity) of a flat protective polymer layer on the accessibility of its functional groups and efficiency of receptor targeting. Two types of receptor surfaces were considered: the surface homogeneously covered with receptors and the surface containing a finite number of receptor sites. We found that multivalent ligands provide a larger density of targeting groups on the periphery of the layer compared to monovalent ligands for the same overall number of targeting groups per polymer layer. Because of their cooperativity in binding, multivalent ligands were also considerably more efficient in binding to both types of receptor surfaces. With an increase of ligand valence the number of functional groups attached to receptors noticeably increases. Short-branched divalent ligands show an especially high cooperativity in binding to closely packed receptors. However, in the case of immobile receptors separated by a finite distance from each other, the average distance between the functional groups belonging to the same short divalent ligand is too small to reach different receptors simultaneously and the receptor binding is less efficient than in the monovalent ligand case. Using a bidisperse protective polymer layer formed by short nonfunctional polymers and long functionalized polymers considerably increases the fraction of functional groups on the periphery of the layer. Simulations of receptor binding confirm the high efficiency of receptor targeting by bidisperse polymer layers, which is achieved by means of larger compressibility and higher capability of the ligands to reach out compared to the corresponding monodisperse layers. The concepts of multivalent ligands and a bidisperse protective polymer layer each have their own advantages which can be combined for an enhanced targeting effect.     

Reversible regulation of protein binding affinity by a DNA machine

We report a DNA machine that can reversibly regulate target binding affinity on the basis of distance-dependent bivalent binding. It is a tweezer-like DNA machine that can tune the spatial distance between two ligands to construct or destroy the bivalent binding. The DNA machine can strongly bind to the target protein when the ligands are placed at an appropriate distance but releases the target when the bivalent binding is disrupted by enlargement of the distance between the ligands. This "capture-release" cycle could be repeatedly driven by single-stranded DNA without changing the ligands and target protein.     

Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin

Multivalent ligand design constitutes an attractive avenue to the inhibition of receptor recognition and other biological events mediated by oligomeric proteins with multiple binding sites. One example is the design of multivalent receptor blockers targeting members of the AB(5) bacterial toxin family. We report here the synthesis and characterization of a pentavalent inhibitor for cholera toxin and Escherichia coli heat-labile enterotoxin. This inhibitor is an advance over the symmetric pentacyclen-derived inhibitor described in our earlier work in that it presents five copies of m-nitrophenyl-alpha-D-galactoside (MNPG) rather than five copies of beta-D-galactose. The approximately 100-fold higher single-site affinity of MNPG for the toxin receptor binding site relative to galactose is found to yield a proportionate increase in the affinity and IC50 measured for the respective pentavalent constructs. We show by dynamic light scattering that inhibition of receptor binding by the pentavalent inhibitor is due to 1:1 inhibitor:toxin association rather than to inhibitor-mediated aggregation. This 1:1 association is in complete agreement with a 1.46 A resolution crystal structure of the pentavalent inhibitor:toxin complex, which shows that the favorable single-site binding interactions of MNPG are retained by the five arms of the 5256 Da pentavalent MNPG-based inhibitor and that the initial segment of the linking groups interacts with the surface of the toxin B pentamer.     

Using covalent dimers of human carbonic anhydrase II to model bivalency in immunoglobulins

This paper describes the development of a new bivalent system comprising synthetic dimers of carbonic anhydrase linked chemically through thiol groups of cysteine residues introduced by site-directed mutagenesis. These compounds serve as models with which to study the interaction of bivalent proteins with ligands presented at the surface of mixed self-assembled monolayers (SAMs). Monovalent carbonic anhydrase (CA) binds to benzenesulfonamide ligands presented on the surface of the SAM with K(d)(surf) = 89 nM. The synthetic bivalent proteins--inspired by the structure of immunoglobulins--bind bivalently to the sulfonamide-functionalized SAMs with low nanomolar avidities (K(d)(avidity,surf) = 1-3 nM); this difference represents a ~50-fold enhancement of bivalent over monovalent association. The paper describes dimers of CA having (i) different lengths of the covalent linker that joined the two proteins and (ii) different points of attachment of the linker to the protein (either near the active site (C133) or distal to the active site (C185)). Comparison of the thermodynamics of their interactions with SAMs presenting arylsulfonamide groups demonstrated that varying the length of the linker between the molecules of CA had virtually no effect on the rate of association, or on the avidity of these dimers with ligand-presenting surfaces. Varying the point of attachment of the linker between monomeric CA's also had almost no effect on the avidity of the dimers, although changing the point of attachment affected the rates of binding and unbinding. These observations indicate that the avidities of these bivalent proteins, and by inference the avidities of structurally similar bivalent proteins such as IgG, are unexpectedly insensitive to the structure of the linker connecting them.     

On the nature of the multivalency effect: a thermodynamic model

A quantitative model is proposed for the analysis of the thermodynamic parameters of multivalent interactions in dilute solutions or with immobilized multimeric receptor. The model takes into account all bound species and describes multivalent binding via two microscopic binding energies corresponding to inter- and intramolecular interactions (Delta G(o)inter and Delta G(o)intra), the relative contributions of which depend on the distribution of complexes with different numbers of occupied binding sites. The third component of the overall free energy, which we call the "avidity entropy" term, is a function of the degeneracy of bound states, Omega(i), which is calculated on the basis of the topology of interaction and the distribution of all bound species. This term grows rapidly with the number of receptor sites and ligand multivalency, it always favors binding, and explains why multivalency can overcome the loss of conformational entropy when ligands displayed at the ends of long tethers are bound. The microscopic parameters and may be determined from the observed binding energies for a set of oligovalent ligands by nonlinear fitting with the theoretical model. Here binding data obtained from two series of oligovalent carbohydrate inhibitors for Shiga-like toxins were used to verify the theory. The decavalent and octavalent inhibitors exhibit subnanomolar activity and are the most active soluble inhibitors yet seen that block Shiga-like toxin binding to its native receptor. The theory developed here in conjunction with our protocol for the optimization of tether length provides a predictive approach to design and maximize the avidity of multivalent ligands.     

Exploring the Limits of Bivalency by DNA‐Based Spatial Screening

Multivalency can facilitate complex formation when monovalent receptor–ligand interactions are weak. However, enhanced binding of two multivalent binding partners should be avoidable, for example when bivalent receptors ought to utilize multimolecular interactions to cross‐link binding partners. We herein report the first systematic study to assess the criteria deciding whether a bivalent system engages in bivalency‐enhanced interactions or cross‐linking. We used DNA‐instructed self‐assembly to arrange the cucurbit[7]uril–adamantane host–guest system in 70–360 Å distance. Measurements and statistical mechanics analyses revealed that the affinity gain is controlled by 1) the distance between recognition modules, 2) the scaffold flexibility, and, importantly, 3) the strength of the monovalent interaction. We show that the bivalency effect can extend beyond 150 Å and discuss how, on the contrary, weak monovalent interactions reduce the concentration threshold for cross‐linking. The findings are of interest for inhibitor design.     

Synthesis and cholera toxin binding properties of a lactose-2-aminothiazoline conjugate

[structure: see text] During the search for improved monovalent ligands for cholera toxin (CT), a new lactose-2-aminothiazoline conjugate was discovered. In a fluorescence binding assay the compound was found to be one of the strongest relatively simple CT ligands to date with a K(d) of 23 microM.     

Design of Monodisperse and Well‐Defined Polypeptide‐Based Polyvalent Inhibitors of Anthrax Toxin

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure–activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide‐based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin‐binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells.     

On the magnitude of the chelate effect for the recognition of proteins by pharmacophores scaffolded by self-assembling oligonucleotides

The simultaneous interaction of the binding moieties of a bidentate ligand on adjacent epitopes of a target protein represents an attractive avenue for the discovery of specific, high-affinity binders. We used short DNA fragments in heteroduplex format to scaffold pairs of binding molecules with defined spatial arrangements. Iminobiotin derivates were coupled either via bifunctional linkers or by using various oligonucleotides, thus allowing monovalent or bivalent binding to streptavidin. We determined the binding affinities of the synthesized constructs in solution. We also investigated the efficiency of recovery of superior bidentate ligands in affinity capture experiments, by using both radioactive counts and DNA microarrays as readouts. This analysis confirmed the suitability of the DNA heteroduplex as a scaffold for the identification of synergistic pairs of binding moieties, capable of a high-affinity interaction with protein targets by virtue of the chelate effect.