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Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity ( K d ) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging. The present work introduces ME mbrane AN chor-assisted nMS (MEAN-nMS) for the detection of low-affinity GBP-GSL complexes. The assay utilizes a membrane anchor, produced by covalent cross-linking of the GBP and a lipid in the membrane, to localize the GBP on the surface and promote GSL binding. Ligands are identified by nMS detection of intact GBP-GSL complexes (MEAN-nMS) or using a catch-and-release (CaR) strategy, wherein GSLs are released from GBP-GSL complexes upon collisional activation and detected (MEAN-CaR-nMS). To establish reliability, a library of purified gangliosides incorporated into nanodiscs was screened against human immune lectins, and the results compared with affinities of the corresponding ganglioside oligosaccharides. Without a membrane anchor, nMS analysis yielded predominantly false negatives. In contrast, all ligands were identified by MEAN-(CaR)-nMS, with no false positives. To highlight the potential of MEAN-CaR-nMS for ligand discovery, a natural library of GSLs was incorporated into nanodiscs and screened against human and viral proteins to uncover elusive ligands. Finally, nMS-based detection of GSL ligands directly from cells is demonstrated. This breakthrough paves the way for shotgun glycomics screening using intact cells.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes in vitro and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs). For many biomolecular complexes, such as protein-protein interactions, this condition is not satisfied. Existing strategies to correct for nonuniform RFs are generally incompatible with static nanoflow ESI (nanoESI) sources, which are typically used for biomolecular interaction studies, thereby significantly limiting the utility of ESI-MS. Here, we introduce slow mixing mode (SLOMO) nanoESI-MS, a direct technique that allows both the RF and affinity ( K d ) for a biomolecular interaction to be determined from a single measurement using static nanoESI. The approach relies on the continuous monitoring of interacting species and their complexes under nonhomogeneous solution conditions. Changes in ion signals of free and bound species as the system approaches or moves away from a steady-state condition allow the relative RFs of the free and bound species to be determined. Combining the relative RF and the relative abundances measured under equilibrium conditions enables the K d to be calculated. The reliability of SLOMO and its ease of use is demonstrated through affinity measurements performed on peptide-antibiotic, protease-protein inhibitor, and protein oligomerization systems. Finally, affinities measured for the binding of human and bacterial lectins to a nanobody, a viral glycoprotein, and glycolipids displayed within a model membrane highlight the tremendous power and versatility of SLOMO for accurately quantifying a wide range of biomolecular interactions important to human health and disease., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Mass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured. In principle, relative affinities can be ranked by combining CaR-ESI-MS data with relative concentrations established by hydrophilic interaction liquid chromatography (HILIC) performed on the fluorophore-labeled glycan library. To validate this approach, as well as the feasibility of performing CaR-ESI-MS directly on labeled glycans, libraries of labeled N -glycans extracted from the human monocytic U937 cells or intestinal tissues were labeled with 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), or procainamide (proA). The libraries were screened against plant and human GBPs with known specificities for α2-3- and α2-6-linked sialosides and quantified by HILIC. Dramatic differences, in some cases, were found for affinity rankings obtained with libraries labeled with different fluorophores, as well as those produced using the combined unlabeled/labeled library approach. The origin of these differences could be explained by differential glycan labeling efficiencies, the impact of specific labels on glycan affinities for the GBPs, and the relative efficiency of release of ligands from GBPs in CaR-ESI-MS. Overall, the results of this study suggest that the 2-AB(CaR-ESI-MS)/2-AB(HILIC) combination provides the most reliable description of the binding specificities of GBPs for N -glycans and is recommended for MS-SG applications.

Carbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking. Here, we introduce a simple but quantitative electrospray ionization mass spectrometry (ESI-MS) method for measuring the kinetics of CAZyme reactions involving glycoprotein substrates. The assay, referred to as center-of-mass (CoM) monitoring (CoMMon), relies on continuous (real-time) monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labeling, or mass spectrum deconvolution. To demonstrate the reliability of CoMMon, we applied the method to the neuraminidase-catalyzed cleavage of N -acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined with CoMMon are in good agreement (initial rates within ≤5%) with results obtained, simultaneously, using an isotopically labeled Neu5Ac internal standard, which enabled the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay was used to measure the kinetics of sialylation of a series of asialo-glycoproteins by a human sialyltransferase. Finally, we show how combining CoMMon and the competitive universal proxy receptor assay enables the relative reactivity of glycoprotein substrates to be quantitatively established.

Interactions between glycosphingolipids (GSLs) on the surfaces of cells and glycan-binding proteins (GBPs) mediate a wide variety of essential and pathological processes. Despite the biological importance of these interactions, the GSL ligands of most GBPs remain to be identified and the mechanisms controlling recognition of GSLs are incompletely understood. Recently, it was suggested that, when present together with high affinity ligands, low affinity GSL ligands can contribute significantly to the binding of GBPs with multiple binding sites through a process called heteromultivalent binding. Here, with goal of directly establishing the existence of heteromultivalent GSL interactions and elucidating the mechanism underlying their formation, we investigated cholera toxin B subunit homopentamer (CTB 5 ) binding to ganglioside mixtures in model membranes (nanodiscs) using native mass spectrometry (MS) and competitive ligand binding. Electrospray ionization (ESI)-MS analysis revealed that the presence of the high affinity ligand GM1 (at substoichiometric amounts relative to binding sites) in the nanodisc promotes GD1b binding to CTB 5 ; no GD1b binding was detected in the absence of GM1. No direct ESI-MS evidence of CTB 5 binding to the other five gangliosides tested, alone or present together with GM1 in the nanodiscs, was observed. Affinity measurements, carried out using the proxy ligand ESI-MS binding assay, confirmed that GD1b binding to CTB 5 is dramatically enhanced (>1000-times higher affinity compared to the GD1b oligosaccharide affinity) when present with GM1. NDs containing GM1 and GM2, GD1a, or GT1b also exhibited enhanced CTB 5 binding, however, the effect was smaller. The results of molecular dynamics simulations performed on ganglioside-containing nanodiscs suggest that the participation of low affinity ligands in heteromultivalent binding with GM1 may be regulated by the positions of the internal Gal-linked Neu5Ac residues of the gangliosides relative to the membrane surface.

Carbohydrate-Active enZymes (CAZymes) are involved in the synthesis, degradation, and modification of carbohydrates. They play critical roles in diverse physiological and pathophysiological processes, have important industrial and biotechnological applications, are important drug targets, and represent promising biomarkers for the diagnosis of a variety of diseases. Measurements of their activities, catalytic pathway, and substrate specificities are essential to a comprehensive understanding of the biological functions of CAZymes and exploiting these enzymes for industrial and biomedical applications. For glycosyl hydrolases a variety of sensitive and quantitative spectrophotometric techniques are available. However, measuring the activity of glycosyltransferases is considerably more challenging. Here, we introduce CUPRA-ZYME, a versatile and quantitative electrospray ionization mass spectrometry (ESI-MS) assay for measuring the kinetic parameters of CAZymes, monitoring reaction pathways, and profiling substrate specificities. The method employs the recently developed competitive universal proxy receptor assay (CUPRA), implemented in a time-resolved manner. Measurements of the hydrolysis kinetics of CUPRA substrates containing ganglioside oligosaccharides by the glycosyl hydrolase human neuraminidase 3 served to validate the reliability of kinetic parameters measured by CUPRA-ZYME and highlight its use in establishing catalytic pathways. Applications to libraries of substrates demonstrate the potential of the assay for quantitative profiling of the substrate specificities glycosidases and glycosyltransferases. Finally, we show how the comparison of the reactivity of CUPRA substrates and glycan substrates present on glycoproteins, measured simultaneously, affords a unique opportunity to quantitatively study how the structure and protein environment of natural glycoconjugate substrates influences CAZyme activity.

Glycan interactions with glycan-binding proteins (GBPs) play essential roles in a wide variety of cellular processes. Currently, the glycan specificities of GBPs are most often inferred from binding data generated using glycan arrays, wherein the GBP is incubated with oligosaccharides immobilized on a glass surface. Detection of glycan-GBP binding is typically fluorescence-based, involving the labeling of the GBP with a fluorophore or with biotin, which binds to fluorophore-labeled streptavidin, or using a fluorophore-labeled antibody that recognizes the GBP. While it is known that covalent labeling of a GBP may influence its binding properties, these effects have not been well studied and are usually overlooked when analyzing glycan array data. In the present study, electrospray ionization mass spectrometry (ESI-MS) was used to quantitatively evaluate the impact of GBP labeling on oligosaccharide affinities and specificities. The influence of three common labeling approaches, biotinylation, labeling with a fluorescent dye and introducing an iodination reagent, on the affinities of a series of human milk and blood group oligosaccharides for a C-terminal fragment of human galectin-3 was evaluated. In all cases labeling resulted in a measurable decrease in oligosaccharide affinity, by as much as 90%, and the magnitude of the change was sensitive to the nature of the ligand. These findings demonstrate that GBP labeling may affect both the absolute and relative affinities and, thereby, obscure the true glycan binding properties. These results also serve to illustrate the utility of the direct ESI-MS assay for quantitatively evaluating the effects of protein labeling on ligand binding., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Electrospray ionization mass spectrometry (ESI-MS) screening of compound libraries against target proteins enables the rapid identification of ligands and measurement of the stoichiometry and affinity of the interactions. However, non-specific association of buffer or salts (added or present as impurities) to the protein ions during gas-phase ion formation can complicate the analysis of ESI-MS data acquired for mixtures of compounds with similar molecular weights. Spectral overlap of ions corresponding to free protein and protein-ligand complexes and their corresponding adducts can hinder the identification of ligands and introduce errors in the measured affinities. Here, we present a straightforward approach, called the sliding window adduct removal method (SWARM), to quantitatively correct ESI mass spectra of low-to-moderate resolution for signal overlap associated with adducts. The method relies on the statistical nature of adduct formation in ESI and the assumption that the distributions of adducts associated with a given protein (free protein and ligand-bound forms) are identical at a given charge state. Analysis of ESI mass spectra measured for protein-oligosaccharide interactions using solutions that produced either low- or high-abundance adducts provides support for this assumption. Implementation of SWARM involves the stepwise subtraction of the adduct signal associated with the detected protein-ligand complexes from the mass spectrum. This is accomplished using the adduct distribution measured for an appropriate reference species (usually free protein). To demonstrate the utility of the method, we applied SWARM to ESI-MS screening data acquired for libraries of oligosaccharides and bifunctional ligands consisting of a sulfonamide moiety linked to human glycan structures. Graphical Abstract.

Glycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes. Comprehensive mapping of glycan interactions is essential to understanding of glycan-mediated biology and can guide the development of new diagnostics and therapeutics. Here, we introduce the competitive universal proxy receptor assay (CUPRA), which combines electrospray ionization mass spectrometry, competitive binding and heterobifunctional glycan-based ligands to give a quantitative high-throughput method for screening glycan libraries against glycan-binding and glycan-processing proteins. Application of the assay to human (siglec-2), plant ( Sambucus nigra and Maackia amurensis lectins) and bacterial (cholera toxin, and family 51 carbohydrate binding module) proteins allowed for the identification of ligands with affinities ( K d ) ≤ 1 mM. The assay is unprecedentedly versatile and can be applied to natural libraries and, when implemented in a time-resolved manner, provides a quantitative measure of the activities and substrate specificity of carbohydrate-active enzymes., Competing Interests: Competing interestsThe authors declare no competing interests.

Human milk oligosaccharides (HMOs) afford many health benefits to breast-fed infants, such as protection against infection and regulation of the immune system, through the formation of non-covalent interactions with protein receptors. However, the molecular details of these interactions are poorly understood. Here, we describe the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening natural libraries of HMOs against lectins. The HMOs in the libraries were first identified based on molecular weights (MWs), ion mobility separation arrival times (IMS-ATs) and collision-induced dissociation (CID) fingerprints of their deprotonated anions. The libraries were then screened against lectins and the ligands identified from the MWs, IMS-ATs and CID fingerprints of HMOs released from the lectin in the gas phase. To demonstrate the assay, four fractions, extracted from pooled human milk and containing ≥35 different HMOs, were screened against a C-terminal fragment of human galectin-3 (hGal-3C), for which the HMOs specificities have been previously investigated, and a fragment of the blood group antigen-binding adhesin (BabA) from Helicobacter pylori, for which the HMO specificities have not been previously established. The structures of twenty-one ligands, corresponding to both neutral and acidic HMOs, of hGal-3C were identified; all twenty-one were previously shown to be ligands for this lectin. The presence of HMO ligands at six other MWs was also ascertained. Application of the assay to BabA revealed nineteen specific HMO structures that are recognized by the protein and HMO ligands at two other MWs. Notably, it was found that BabA exhibits broad specificity for HMOs, and recognizes both neutral HMOs, including non-fucosylated ones, and acidic HMOs. The results of competitive binding experiments indicate that HMOs can interact with BabA at previously unknown binding sites. The affinities of eight purified HMOs for BabA were measured by ESI-MS and found to be in the 10 3 M -1 to 10 4 M -1 range.

Using amphiphilic cyclodextrin as a scaffold, the first class of P K -glycoconjugates capable of high avidity binding to both Stx1 and Stx2 toxins in solid-phase assay formats is reported. The generated glycomicroarray effectively mimics the plasma membrane surface while discriminating binding of the two Stx toxins, with unprecedented affinity to Stx2.

The affinities of thirty-two free human milk oligosaccharides (HMOs) for four human galectin proteins, a stable mutant of hGal1 (hGal-1), a C-terminal fragment of hGal-3 (hGal-3C), hGal-7, and an N-terminal fragment of hGal-9 (hGal-9N), were measured using electrospray ionization mass spectrometry (ESI-MS). The binding data show that each of the four galectins recognize the majority of the HMOs tested (hGal-1 binds thirty-two HMOs, hGal-3C binds twenty-six, hGal-7 binds thirty-one, and hGal-9N binds twenty-six). Twenty-five of the HMOs tested bind all four galectins, with affinities ranging from 10 3 to 10 5 M -1 . The reliability of the ESI-MS assay for quantifying the affinities of HMOs for lectins was established from the agreement found between the ESI-MS data and affinities of a small number of HMOs for hGal-1, hGal-3C, and hGal-7 measured by isothermal titration calorimetry (ITC). Comparison of the relative affinities (of 14 HMOs) measured by ESI-MS with the reported specificities of hGal-1, hGal-3, hGal-7, and hGal-9 for these same HMOs established using the shotgun human milk glycan microarray (HM-SGM-v2) showed fair-to-poor correlation, with evidence of false positives and false negatives in the microarray data. The results of this study suggest that HMO specificities of lectins established using microarrays may not accurately reflect their true HMO-binding properties and that the use of "in solution" assays such as ESI-MS and ITC is to be preferred.

[This corrects the article DOI: 10.1039/C5SC03856A.].

In this manuscript, we describe modification of Cys-residues in peptides and proteins in aqueous solvents via aromatic nucleophilic substitution (S N Ar) with perfluoroarenes (fAr). Biocompatibility of this reaction makes it attractive for derivatization of proteins and peptide libraries comprised of 20 natural amino acids. Measurement of the reaction rates for fAr derivatives by 19 F NMR with a model thiol donor (β-mercaptoethanol) in aqueous buffers identified decafluoro-diphenylsulfone ( DFS ) as the most reactive S N Ar electrophile. Reaction of DFS with thiol nucleophiles is >100 000 faster than analogous reaction of perfluorobenzene; this increase in reactivity enables application of DFS at low concentrations in aqueous solutions compatible with biomolecules and protein complexes irreversibly degraded by organic solvents ( e.g. , bacteriophages). DFS forms macrocycles when reacted with peptides of the general structure X n -Cys-X m -Cys-X l , where X is any amino acid and m = 1-15. It formed cyclic peptides with 6 peptide hormones-oxytocin, urotensin II, salmon calcitonin, melanin-concentrating hormone, somatostatin-14, and atrial natriuretic factor (1-28) as well as peptides displayed on M13 phage. Rates up to 180 M -1 s -1 make this reaction one of the fastest Cys-modifications to-date. Long-term stability of macrocycles derived from DFS and their stability toward oxidation further supports DFS as a promising method for modification of peptide-based ligands, cyclization of genetically-encoded peptide libraries, and discovery of bioactive macrocyclic peptides.

In vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc., where [X]4 denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide-WIVP, with Kd = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide-GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other in vitro display technologies such as bacteriophage T7 or mRNA display.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins—ConA, LcH, and PSA—that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.

We describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivatives with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramolecular ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis. We characterized the reaction by both NMR and UV spectroscopy and determined that the rate-determining step of the process is formation of a Schiff base, which is followed by rapid intramolecular ring closure. The relationship between apparent rate constant and pH suggests that a protonated benzamidoxime acts as an internal general acid in Schiff-base formation. The reaction is accelerated by substituents in the aromatic ring that increase the basicity of the aromatic amine. The rate of up to 40 M(-1) s(-1) between an electron-rich aldehyde and 5-methoxy-ABAO (PMA), which was observed at pH 4.5, places this reaction among the fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO derivative reaches completion in 1 h at pH 4.5. Finally, the product of reaction, dihydroquinazoline derivative, shows fluorescence at 490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive probes based on ABAO framework.

Copovidone, a copolymer of vinyl acetate and N-vinyl-2-pyrrolidone, was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and after deacetylation the polymer was functionalized by introduction of amino, azide, and alkyne pendant groups to allow attachment of glycans and peptide. Candida albicans β-mannan trisaccharides 1 and 2 and M. tuberculosis arabinan hexasaccharide 3 with appropriate tethers were conjugated to the polymers by squarate or click chemistry. C. albicans T-cell peptide 4 bearing a C-terminal ε-azidolysine was also conjugated to copovidone by click chemistry. The resulting conjugates provide convenient non-protein-based antigens that are readily adsorbed on ELISA plates, and display excellent characteristics for assay of antibody binding to the haptenic group of interest. Copovidone and BSA glycoconjugates exhibited similar adsorption characteristics when used to coat ELISA plates, and both conjugates were optimal when used as coating solutions at low nanogram/mL concentrations. Provided that the copovidone conjugated glycan is stable to acid, assay plates can be easily processed for reuse at least three times without detectable variation or degradation in ELISA readout.

Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library. We ligated a photoresponsive azobenzene core to a random CX7C peptide library displayed on the coat protein of M13 phage. A one-pot alkylation/reduction of the cysteines yielded a photoresponsive library of random heptapeptide macrocycles with over 2 × 10(8) members. We characterized the reaction on-phage and optimized the yield of the modifications in phage libraries. Screening of the library against streptavidin yielded three macrocycles that bind to streptavidin in the dark and cease binding upon irradiation with 370 nm light. All ligands restored their binding properties upon thermal relaxation and could be turned ON and OFF for several cycles. We measured dissociation constants, Kd, by electrospray ionization mass spectrometry (ESI-MS) binding assay. For ligand ACGFERERTCG, the Kd of cis and trans isomers differed by 22-fold; an incomplete isomerization (85%), however, resulted in the apparent difference of 4.5-fold between the dark and the irradiated state. We anticipate that the selection strategy described in this report can be used to find light-responsive ligands for many targets that do not have known natural ligands.

A new microtiter-plate-based method for the rapid generation and evaluation of focused compound libraries was developed and applied to screening ligand analogues for the E. coli Shiga-like toxin Stx2a. The method is general, it mitigates the masking of intrinsic affinity gains by multivalency and enables the discovery of potential hits when starting from ligands that exhibit extremely low affinity with proteins that depend on multivalency for their function., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

A convenient scaffold based on poly(N-vinyl-2-pyrrolidone-co-vinyl alcohol) is proposed for presenting ligands in multivalent format. This amphiphilic polymer supports synthesis of conjugates in both organic and aqueous media, permits enzymatic processing of the ligand precursor, and, finally, offers a choice of formats for evaluation of biological activity either as a soluble inhibitor or as a capture reagent after deposition on a hydrophobic surface or standard microtiter plates.

Hemolytic uremic syndrome is a potentially fatal complication of food poisoning caused by Escherichia coli O157:H7, especially those strains that produce the Stx2 Shiga toxin. Multivalent inhibitors based on the P(k) trisaccharide are most effective against Stx1 the less dangerous of the two Shiga toxins. Inhibitors containing a terminal 2-acetamido-2-deoxy-α-d-galactopyranosyl residue in place of the terminal α-d-galactopyranosyl residue of P(k) trisaccharide have been shown to exhibit preferential binding to Stx2. A multivalent heterobifunctional P(k) analog containing 2-acetamido-2-deoxy-α-d-galactopyranose has been synthesized in a format that facilitates the ablation of toxin activity via supramolecular complex formation between Stx and the endogenous protein, Human serum amyloid P component (HuSAP)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Noroviruses (NoVs), the major cause of viral acute gastroenteritis, recognize histo-blood group antigens (HBGAs) as receptors or attachment factors. To gain a deeper understanding of the interplay between NoVs and their hosts, the affinities of recombinant P dimers (P₂'s) of a GII.4 NoV (VA387) to a library of 41 soluble analogs of HBGAs were measured using the direct electrospray ionization mass spectrometry assay. The HBGAs contained the A, B, H and Lewis epitopes, with variable sizes (2-6 residues) and different types (1-6). The results reveal that the P₂'s exhibit a broad specificity for the HBGAs and bind to all of the oligosaccharides tested. Overall, the affinities are relatively low, ranging from 400 to 3000 M⁻¹ and are influenced by the chain type: 3 > 1 ≈ 2 ≈ 4 ≈ 5 ≈ 6 for H antigens; 6 > 1 ≈ 3 ≈ 4 ≈ 5 > 2 for A antigens; 3 > 1 ≈ 4 ≈ 5 ≈ 6 > 2 for B antigens, but not by chain length. The highest-affinity ligands are B type 3 (3000 ± 300 M⁻¹) and A type 6 (2350 ± 60 M⁻¹). While the higher affinity to the type 3 H antigen was previously observed, preferential binding to the types 6 and 3 antigens with A and B epitopes, respectively, has not been previously reported. A truncated P domain dimer (lacking the C-terminal arginine cluster) exhibits similar binding. The central-binding motifs in the HBGAs were identified by molecular-docking simulations.

Inhibition of AB(5)-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin's pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB(5) complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor's activity.

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(β1-4)Glc, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc and Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3 )M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.

Noroviruses attach to their host cells through histo blood group antigens (HBGAs), and compounds that interfere with this interaction are likely to be of therapeutic or diagnostic interest. It is shown that NMR binding studies can simultaneously identify and differentiate the site for binding HBGA ligands and complementary ligands from a large compound library, thereby facilitating the design of potent heterobifunctional ligands. Saturation transfer difference (STD) NMR experiments, spin-lock filtered NMR experiments, and interligand NOE (ILOE) experiments in the presence of virus-like particles (VLPs), identified compounds that bind to the HBGA binding site of human norovirus. Based on these data two multivalent prototype entry-inhibitors against norovirus infection were synthesized. A surface plasmon resonance based inhibition assay showed avidity gains of 1000 and one million fold over a millimolar univalent ligand. This suggests that further rational design of multivalent inhibitors based on our strategy will identify potent entry-inhibitors against norovirus infections., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Structural pre-organization of the multivalent ligands is important for successful interaction with multimeric proteins. Polymer-based heterobifunctional ligands that contain pendant groups prearranged into heterodimers can be used to probe the active site and surrounding area of the receptor. Here we describe the synthesis and activities of a series of galactose conjugates on polyacrylamide and dextran. Conjugation of a second fragment resulted in nanomolar inhibitors of cholera toxin, while the galactose-only progenitors showed no detectable activity.

The tether employed to covalently attach β-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1 → 2)-β-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.

We report the synthesis and in vitro evaluation of a multivalent homing device, a polymer which contains preordered pendant groups with dual specificity, a trisaccharide moiety, which is specific for the siglec CD22, and an antibody specific hapten, nitrophenol. The device efficiently attracts antihapten IgM to the surface of human lymphoma B cells as well as to CD22-conjugated magnetic beads by mediating the formation of a ternary complex on the surface of the target.

A protective β-mannan trisaccharide epitope from the Candida albicans cell wall phosphomannan has been synthesized and activated for copolymerization with acrylamide. The resulting glycopolymer displayed 33 trisaccharide haptens and was derivatized for conjugation to the immunogenic carrier protein, chicken serum albumin. The resulting conjugate achieves a high degree of oligosaccharide substitution while limiting the sites of substitution on the protein. The murine immune response against this conjugate was compared with the response to a trisaccharide-tetanus toxoid conjugate vaccine. The glycopolymer was shown to induce a more robust immune response with higher trisaccharide-specific antibody titers and with a significantly larger proportion of responding mice developing antibodies that bound the target, native cell wall antigen of C. albicans.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi-step process culminating in the intimate adherence of the organisms to the host's intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle-forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin-like properties, which enables BFP to initially tether EPEC to N-acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc-bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc-BSA-induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.

CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells. Surprisingly, anti-NP antibodies of lower valency, IgA (n = 4) and IgG (n = 2), were also found to drive complex formation, though with lower avidity. Ligands bearing alternate linkers of variable length and structure were constructed to establish the importance of a minimal length requirement, and versatility in the structural requirement. We show that the ligand drives assembly of IgM complexes exclusively on the surface of B cells and not other classes of white blood cells that do not express CD22, which lends itself to the possibility of targeting B cells in certain hematopoietic malignancies.

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.

We previously reported that the bundle-forming pilus-mediated localized adherence of enteropathogenic Escherichia coli to HEp-2, T84, and Caco-2 cells is inhibited by N-acetyllactosamine neoglycoconjugates. The results presented here extend this observation to the epithelium of biopsy specimens obtained from the human adult duodenum, terminal ileum, and colon.

In a previous article, the authors reported that exposing wild-type enteropathogenic Escherichia coli (EPEC) to chemically synthesized N-acetyllactosamine glycosides covalently coupled to BSA (LacNAc-BSA) inhibited localized adherence (LA) by these organisms and also caused them to lose their bundle-forming pili (BFP), the filamentous surface appendages responsible for their LA phenotype. This effect has now been further investigated by screening a panel of LacNAc-BSA-related glycosides for their ability to inhibit EPEC LA, which revealed that LacNAc-BSA retained its status as the most effective inhibitor of EPEC LA. It was also shown that LacNAc-BSA did not cause the loss of BFP in an EPEC strain containing a non-polar mutation in the bfpF gene and, as a consequence, unable to retract its BFP. LacNAc-BSA also effectively inhibited LA of the bfpF mutant EPEC. Taken together, these observations suggest that, as well as triggering BfpF-mediated BFP retraction, LacNAc-BSA likely functions as a competitive inhibitor of EPEC binding to LacNAc-related receptors on host cells. Moreover, transmission electron microscopy revealed that LacNAc conjugated to gold nanoparticles bound specifically to BFP. This observation indicated that either the major BFP structural subunit (BfpA) itself or, possibly, an accessory protein co-assembled with BfpA into the BFP filaments, contains a LacNAc-specific EPEC adhesin. The results suggest a mechanism whereby the initial binding of EPEC to LacNAc-like receptors on host cells triggers BfpF-mediated BFP retraction. This could then expedite the intimate adherence phase of the multi-step EPEC colonization process by drawing the organisms closer to the host-cell plasma membrane.

Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP-protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP-BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP-protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.

[reaction: see text] The first example of a multivalent heterofunctional inhibitor-adaptor, called "BAIT", is described. This multivalent inhibitor-adaptor is able to capture a "target" receptor (Shiga toxin) through its recognition of one ligand of a heterobivalent headgroup while the other ligand binds to an endogenous "trap" protein (serum amyloid P component, SAP). BAIT showed markedly enhanced inhibition of toxin activity. An efficient synthesis of this multivalent cluster containing heterobifunctional ligands was accomplished by chemical and chemoenzymatic approaches.

A comprehensive series of solution and crystallographic studies reveal how simple, achiral, bivalent ligands of the cyclic pyruvate of glycerol promote face-to-face complex formation of the pentraxin, serum amyloid P component (SAP) into decamers. SAP, a protein of the human innate immune system, is universally present in amyloids, including cerebral amyloid deposits found in the brain of Alzheimer disease patients. Removal of SAP through a specific aggregation mechanism mediated by multivalent ligands appears to provide therapeutic benefit in the progression of this disease. Crystallographic studies reveal that in our novel series of ligands only the methyl and carboxylate moieties of the pyruvate ketal directly interact with the protein, but the geometric constraints imposed by the tether dictate which of two chair conformations are adopted by the pyruvate dioxane ring. Solution studies, as interpreted through a simple thermodynamic model, account for the distribution of pentameric and decameric bound states at different ligand concentrations and indicate that differences in the flexibility of the tether determine the geometry and stability of the specific aggregates formed between SAP and two different bivalent ligands. The factors affecting the design of ligands promoting face-to-face protein dimerization as well as potential biological implications are discussed.

Purpose: To determine the effect of tether length on bone mineral affinity of fetuin-bisphosphonate conjugates., Methods: 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the lysine residues of fetuin by using five different crosslinkers that varied in length. Both the conjugation efficiency (i.e., the number of aminoBPs per protein) as well as molecular dynamics modeling of the resulting conjugates were assessed. Furthermore, the in vitro and in vivo bone mineral affinity of the conjugates were compared to one another., Results: The tethers, whose extended lengths varied from 5.7 to approximately 136 A, were effective in conjugating aminoBP onto fetuin. Molecular dynamics modeling revealed an inverse relationship between tether length and the maximal radial density of the pendent ligand. The capacity of the conjugates to bind to various bone matrices in vitro differed significantly, as aminoBPs tethered onto fetuin via shorter cross-linkers afforded a superior affinity for various mineral matrices than those tethered via longer cross-linkers. Results from the in vivo mineral implantation studies corroborated the in vitro findings, as higher binding was achieved with the shorter conjugates. Thus, the binding capacities of the conjugates paralleled the maximal radial densities of the pendent ligands., Conclusions: The use of any of the chosen cross-linkers was feasible in conjugating aminoBP onto fetuin. Maximal mineral binding of the fetuin-aminoBP conjugates, however, was typically achieved using the shorter cross-linkers.

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.

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.

The therapeutic potential of 2 soluble multivalent receptor-based inhibitors of Shiga toxin (Stx) 1 and Stx2 was determined in mice. One of these, Starfish, protected mice when it was injected subcutaneously in admixture with a lethal dose of Stx1 but not Stx2. Starfish also reduced the distribution of (125)I-Stx1 but not (125)I-Stx2 to the murine kidney and brain. A modified version of Starfish, called "Daisy," in which the Stx alpha Gal(1,4)beta Gal(1,4)beta Glc receptors were installed on the core glucose structure via a modified tethering strategy, protected mice against both Stx1 and Stx2. Daisy also protected streptomycin-treated mice from Escherichia coli O91:H21 and did not interfere with the ability of the murine immune system to produce Stx-specific protective antibodies. These results extend the possibility of using soluble carbohydrate-based receptor inhibitors to prevent Stx-mediated complications arising from infections with enterohemorrhagic E. coli serotypes.