Multivalent Scaffolds Research Articles

Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection invitro and invivo.

Inhibition of influenza A virus infection by multivalent sialic acid inhibitors preventing viral hemagglutinin binding to host cells of the respiratory tract is a promising strategy. However, optimal geometry and optimal ligand presentation on multivalent scaffolds for efficient inhibition both invitro and invivo application are still unclear. Here, by comparing linear and dendritic polyglycerol sialosides (LPGSA and dPGSA) we identified architectural requirements and optimal ligand densities for an efficient multivalent inhibitor of influenza virus A/X31/1 (H3N2). Due to its large volume, the LPGSA at optimal ligand density sterically shielded the virus significantly better than the dendritic analog. A statistical mechanics model rationalizes the relevance of ligand density, morphology, and the size of multivalent scaffolds for the potential to inhibit virus-cell binding. Optimized LPGSA inhibited virus infection at IC50 in the low nanomolar nanoparticle concentration range and also showed potent antiviral activity against two avian influenza strains A/Mallard/439/2004 (H3N2) and A/turkey/Italy/472/1999 (H7N1) post infection. In vivo application of inhibitors clearly confirmed the higher inhibition potential of linear multivalent scaffolds to prevent infection. The optimized LPGSA did not show any acute toxicity, and was much more potent than the neuraminidase inhibitor oseltamivir carboxylate invivo. Combined application of the LPGSA and oseltamivir carboxylate revealed a synergistic inhibitory effect and successfully prevented influenza virus infection in mice.

V1V2 multivalent scaffolds induce focused antibody responses with functional activity, prolonged durability, and modest neutralization potency

Abstract The V1V2 region of HIV-1 gp120 is vulnerable to a group of broadly neutralizing monoclonal antibodies (bnMAbs), and RV144 vaccinees developed strongly binding Abs to this region that correlated with a reduced risk of infection. This region of Env is now recognized as an important vaccine target, notwithstanding its relatively poorly immunogenic epitopes. We hypothesized that presenting V1V2 in multivalent scaffolds could improve immunogenicity and structurally constraining the epitopes could induce a more focused immune response in animal models. We designed of a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds. Here, we report results of immunogenicity studies in nonhuman primates (NHP) with 2 different V1V2 trimeric scaffolds. Our experimental approach in NHP consisted of 3 DNA and protein co-immunizations and a single protein boost. The DNA components consisted of 50μg of ZM109 gp120 plasmid delivered by gene gun with a simultaneous intramuscular injection of 100 μg of V1V2 scaffold protein immunogens. NHP immune responses were specific for V1V2 conformational epitopes with cross-reactive binding to heterologous gp120s and were functionally active in phagocytosis assays. Notably, V1V2 binding Ab responses were detectable 2 years after immunizations ceased. Neutralizing Abs developed in vaccinated NHP to Tier 1A and 1B, Clade B and Clade C viruses. The V1V2 structurally constrained scaffolds show promise as novel and highly effective at inducing functional antibody responses specifically targeted to the epitopes presented. Our results advance pre-clinical data in support of developing V1V2 vaccine candidates despite the structural complexity of the region. Funding: P01AI100151-03

Preparation of peptide-MHC and T-cell receptor dextramers by biotinylated dextran doping.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I-mediated antigen presentation.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

ABSTRACTBroadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches.

Characterizing Three Component Heteromeric Complexes Mediated by Otoferlin, a Multivalent Membrane Fusion Protein, Using a Novel Single-Molecule Assay

Multidomain proteins reside at the heart of cellular signaling, where they can act as multivalent scaffolds for the assembly of protein complexes and as ligand-dependent effectors of signal transduction. Despite their prevalence, it is unclear how multidomain proteins simultaneously interact with multiple clients, and how each domain contributes to the overall activity of the protein. Challenges, including the large size and low solubility have largely limited studies to smaller truncated forms of these proteins. Although more tractable, the relevance of studies on truncated proteins to our understanding of the full-length protein is questionable. Otoferlin, which consists of 6 consecutive C2 domains, serves an example. Expressed almost exclusively in vertebrate sensory hair cells, otoferlin is a 240 kDa transmembrane protein hypothesized to act as the calcium sensor for neurotransmitter release at hair cell ribbon synapses during the encoding of sound. While studies have suggested that some C2 domains of otoferlin may associate with SNARE proteins and the voltage gated calcium channel CaV1.3, no study has quantitatively characterized multivalent and concomitant binding properties of multi-domain or full-length otoferlin owing to its size and solubility. Thus, it remains unclear whether otoferlin can bind these proteins simultaneously and more generally. To address this limitation, we have developed a generally applicable real-time single-molecule colocalization binding titration assay which in combination with single-molecule photobleaching can resolve the binding multivalencies, stoichiometries, and binding affinities of large full-length multidomain proteins enriched from mammalian cell culture. Using this methodology, we conclude that one otoferlin molecule may bind both up to 4 Cav1.3 and 4 SNARE proteins simultaneously, and that physiologically relevant calcium concentrations alter the stoichiometry and affinity. Our results motivate a model in which otoferlin acts as a scaffolding/priming protein that localizes SNARE proteins and the calcium channel in close proximity to synchronize calcium influx and exocytosis.

Externally controllable glycan presentation on nanoparticle surfaces to modulate lectin recognition.

Nature dynamically controls carbohydrate expression on cells rather than static presentation. Here we report synthetic glycosylated nanoparticles that contain polymeric 'gates' to enable external control (via temperature changes) of glycan surface expression, as an alternative to enzymatic control in nature. This approach offers a new dynamic multivalent scaffold for glycan recognition.

Impact of Dendrimer Terminal Group Chemistry on Blockage of the Anthrax Toxin Channel: A Single Molecule Study.

Nearly all the cationic molecules tested so far have been shown to reversibly block K+ current through the cation-selective PA63 channels of anthrax toxin in a wide nM–mM range of effective concentrations. A significant increase in channel-blocking activity of the cationic compounds was achieved when multiple copies of positively charged ligands were covalently linked to multivalent scaffolds, such as cyclodextrins and dendrimers. Even though multivalent binding can be strong when the individual bonds are relatively weak, for drug discovery purposes we often strive to design multivalent compounds with high individual functional group affinity toward the respective binding site on a multivalent target. Keeping this requirement in mind, here we perform a single-channel/single-molecule study to investigate kinetic parameters of anthrax toxin PA63 channel blockage by second-generation (G2) poly(amido amine) (PAMAM) dendrimers functionalized with different surface ligands, including G2-NH2, G2-OH, G2-succinamate, and G2-COONa. We found that the previously reported difference in IC50 values of the G2-OH/PA63 and G2-NH2/PA63 binding was determined by both on- and off-rates of the reversible dendrimer/channel binding reaction. In 1 M KCl, we observed a decrease of about three folds in and a decrease of only about ten times in with G2-OH compared to G2-NH2. At the same time for both blockers, and increased dramatically with transmembrane voltage increase. PAMAM dendrimers functionalized with negatively charged succinamate, but not carboxyl surface groups, still had some residual activity in inhibiting the anthrax toxin channels. At 100 mV, the on-rate of the G2-succinamate binding was comparable with that of G2-OH but showed weaker voltage dependence when compared to G2-OH and G2-NH2. The residence time of G2-succinamate in the channel exhibited opposite voltage dependence compared to G2-OH and G2-NH2, increasing with the cis-negative voltage increase. We also describe kinetics of the PA63 ion current modulation by two different types of the “imperfect” PAMAM dendrimers, the mixed-surface G2 75% OH 25% NH2 dendrimer and G3-NH2 dendron. At low voltages, both “imperfect” dendrimers show similar rate constants but significantly weaker voltage sensitivity when compared with the intact G2-NH2 PAMAM dendrimer.

Tri- and tetravalent mannoclusters cross-link and aggregate BC2L-A lectin from Burkholderia cenocepacia

The opportunistic Gram-negative bacterium Burkholderia cenocepacia causes lethal infections in cystic fibrosis patients. Multivalent mannoside derivatives were prepared as potential inhibitors of lectin BC2L-A, one of the virulence factors deployed by B. cenocepacia in the infection process. An (α1→2)-thio-linked mannobioside mimic bearing an azide functionalized aglycon was conjugated to different multivalent scaffolds such as propargylated calix[4]arenes, methyl gallate and pentaerythritol by azide-alkyne 1,3-dipolar cycloaddition. The interaction between the glycoclusters and the mannose binding BC2L-A lectin from B. cenocepacia was examined by isothermal microcalorimetry, surface plasmon resonance, inhibition of yeast agglutination and analytical ultracentrifugation.

Mapping ubiquitination sites of S. cerevisiae Mcm10

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.

Pathogen Inhibition by Multivalent Ligand Architectures.

Interfacial multivalent interactions at pathogen-cell interfaces can be competitively inhibited by multivalent scaffolds that prevent pathogen adhesion to the cells during the initial stages of infection. The lack of understanding of complex biological systems makes the design of an efficient multivalent inhibitor a toilsome task. Therefore, we have highlighted the main issues and concerns associated with blocking pathogen at interfaces, which are dependent on the nature and properties of both multivalent inhibitors and pathogens, such as viruses and bacteria. The challenges associated with different cores or carrier scaffolds of multivalent inhibitors are concisely discussed with selected examples.

Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease.

Finding the Right Candidate for the Right Position: A Fast NMR-Assisted Combinatorial Method for Optimizing Nucleic Acids Binders.

Development of strong and selective binders from promiscuous lead compounds represents one of the most expensive and time-consuming tasks in drug discovery. We herein present a novel fragment-based combinatorial strategy for the optimization of multivalent polyamine scaffolds as DNA/RNA ligands. Our protocol provides a quick access to a large variety of regioisomer libraries that can be tested for selective recognition by combining microdialysis assays with simple isotope labeling and NMR experiments. To illustrate our approach, 20 small libraries comprising 100 novel kanamycin-B derivatives have been prepared and evaluated for selective binding to the ribosomal decoding A-Site sequence. Contrary to the common view of NMR as a low-throughput technique, we demonstrate that our NMR methodology represents a valuable alternative for the detection and quantification of complex mixtures, even integrated by highly similar or structurally related derivatives, a common situation in the context of a lead optimization process. Furthermore, this study provides valuable clues about the structural requirements for selective A-site recognition.

BSA/HSA ratio modulates the properties of Ca2+-induced cold gelation scaffolds

An effective tissue engineering approach requires adjustment according to the target tissue to be engineered. The possibility of obtaining a protein-based formulation for the development of multivalent tunable scaffolds that can be adapted for several types of cells and tissues is explored in this work. The incremental substitution of bovine serum albumin (BSA) by human serum albumin (HSA), changing the scaffolds’ hydrophilic/hydrophobic ratio, on a previously optimized scaffold formulation resulted in a set of uniform porous scaffolds with different physical properties and associated cell proliferation profile along time. There was a general trend towards an increase in hydrophilicity, swelling degree and in vitro degradation of the scaffolds with increasing replacement of BSA by HAS. The set of BSA/HSA scaffolds presented distinct values for the storage (elastic) modulus and loss factor which were similar to those described for different native tissues such as bone, cartilage, muscle, skin and neural tissue. The preferential adhesion and proliferation of skin fibroblasts on the BSA25%HSA75% and HSA100% scaffolds, as predicted by their viscoelastic properties, demonstrate that the BSA/HSA scaffold formulation is promising for the development of scaffolds that can be tuned according to the tissue to be repaired and restored.

FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2 +/PKA crosstalk

Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca2+]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium “switch” that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca2+ and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca2+/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca2+ and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may represent an important paradigm for the regulation of cellular signaling networks.

Multivalent Inhibitors of Channel-Forming Bacterial Toxins.

Rational design of multivalent molecules represents a remarkable modern tool to transform weak non-covalent interactions into strong binding by creating multiple finely-tuned points of contact between multivalent ligands and their supposed multivalent targets. Here, we describe several prominent examples where the multivalent blockers were investigated for their ability to directly obstruct oligomeric channel-forming bacterial exotoxins, such as the pore-forming bacterial toxins and B component of the binary bacterial toxins. We address problems related to the blocker/target symmetry match and nature of the functional groups, as well as chemistry and length of the linkers connecting the functional groups to their multivalent scaffolds. Using the anthrax toxin and AB5 toxin case studies, we briefly review how the oligomeric toxin components can be successfully disabled by the multivalent non-channel-blocking inhibitors, which are based on a variety of multivalent scaffolds.

Oriented Immobilized Sialyloligo-macroligand Microarray.

Silaic acid is the most common terminal glycan on cell surface glycoproteins and glycolipids, involving in many biological processes. Studying interactions between multivalent sialic acid scaffolds and proteins binding to it will give incredible information in understanding the biological process the sialic acid is involved in. Here we describe chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications.

Open-resorcinarenes, a new family of multivalent scaffolds.

A new family of multivalent ligand platforms, the open-resorcinarenes, has been prepared in a straightforward two-step reaction. Modification of the core gives a range of topologically diverse scaffolds; functionalisation confirms the versatility of this approach, as shown through the formation of an octacalixarene array.

Micelles from self-assembled double-hydrophilic PHEMA-glycopolymer-diblock copolymers as multivalent scaffolds for lectin binding

We introduce a novel double-hydrophilic hydroxyethylmethacrylate (HEMA) based diblock glycopolymer which self-assembles into homogeneous spherical micellar structures in water.

Modulation of Innate Immune Responses via Covalently Linked TLR Agonists

We present the synthesis of novel adjuvants for vaccine development using multivalent scaffolds and bioconjugation chemistry to spatially manipulate Toll-like receptor (TLR) agonists. TLRs are primary receptors for activation of the innate immune system during vaccination. Vaccines that contain a combination of small and macromolecule TLR agonists elicit more directed immune responses and prolong responses against foreign pathogens. In addition, immune activation is enhanced upon stimulation of two distinct TLRs. Here, we synthesized combinations of TLR agonists as spatially defined tri- and di-agonists to understand how specific TLR agonist combinations contribute to the overall immune response. We covalently conjugated three TLR agonists (TLR4, 7, and 9) to a small molecule core to probe the spatial arrangement of the agonists. Treating immune cells with the linked agonists increased activation of the transcription factor NF-κB and enhanced and directed immune related cytokine production and gene expression beyond cells treated with an unconjugated mixture of the same three agonists. The use of TLR signaling inhibitors and knockout studies confirmed that the tri-agonist molecule activated multiple signaling pathways leading to the observed higher activity. To validate that the TLR4, 7, and 9 agonist combination would activate the immune response to a greater extent, we performed in vivo studies using a vaccinia vaccination model. Mice vaccinated with the linked TLR agonists showed an increase in antibody depth and breadth compared to mice vaccinated with the unconjugated mixture. These studies demonstrate how activation of multiple TLRs through chemically and spatially defined organization assists in guiding immune responses, providing the potential to use chemical tools to design and develop more effective vaccines.

Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding.

Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314–324) and E2 (residues 412–423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.