Multivalent Receptor Research Articles

Multivalent insulin receptor activation using insulin-DNA origami nanostructures.

Insulin binds the insulin receptor (IR) and regulates anabolic processes in target tissues. Impaired IR signalling is associated with multiple diseases, including diabetes, cancer and neurodegenerative disorders. IRs have been reported to form nanoclusters at the cell membrane in several cell types, even in the absence of insulin binding. Here we exploit the nanoscale spatial organization of the IR to achieve controlled multivalent receptor activation. To control insulin nanoscale spatial organization and valency, we developed rod-like insulin-DNA origami nanostructures carrying different numbers of insulin molecules with defined spacings. Increasing the insulin valency per nanostructure markedly extended the residence time of insulin-DNA origami nanostructures at the receptors. Both insulin valency and spacing affected the levels of IR activation in adipocytes. Moreover, the multivalent insulin design associated with the highest levels of IR activation also induced insulin-mediated transcriptional responses more effectively than the corresponding monovalent insulin nanostructures. In an in vivo zebrafish model of diabetes, treatment with multivalent-but not monovalent-insulin nanostructures elicited a reduction in glucose levels. Our results show that the control of insulin multivalency and spatial organization with nanoscale precision modulates the IR responses, independent of the insulin concentration. Therefore, we propose insulin nanoscale organization as a design parameter in developing new insulin therapies.

On the multivalency of large-ring cyclodextrins

The inclusion complexes of several large-ring cyclodextrins with a number of monovalent ligands (five or six adamantane molecules; CDn/mADA; n = 11, 12, 13, 14, 21, 26; m = 5 (for n = 11 to 14) or 6 (for n = 21, 26)) were examined with molecular dynamics simulations. The results demonstrate the high affinity of the LR-CDs to accommodate in their cavities this hydrophobic test particle. Most of the simulation time two guest molecules associate with the CD11 macrocycle. Two to four guest molecules are included in the cavities of CD12, CD13 and CD14 for the total of about 50% to 75% of the simulation time. Higher order associates of CD21 and CD26 with three to five adamantane substrates, comprise more than 40.0% of the snapshots taken from the simulation trajectories, and they still have remaining unoccupied binding sites that could accommodate even more adamantane molecules. Cluster analyses were performed with the k-mean and the bottom up agglomerative hierarchical methods. These LR-CDs, with theirs more than one docking sites are suitable candidates as multivalent receptors for specifically designed multivalent ligands.

Multivalent lectin-carbohydrate interactions: Energetics and mechanisms of binding.

The biological signaling properties of lectins, which are carbohydrate-binding proteins, are due to their ability to bind and cross-link multivalent glycoprotein receptors on the surface of normal and transformed cells. While the cross-linking properties of lectins with multivalent carbohydrates and glycoproteins are relatively well understood, the mechanisms of binding of lectins to multivalent glycoconjugates are less well understood. Recently, the thermodynamics of binding of lectins to synthetic clustered glycosides, a multivalent globular glycoprotein, and to linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules. Importantly, the mechanism of binding of lectins to mucins is similar to that for a variety of protein ligands binding to DNA. Recent analysis also shows that high-affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects which facilitate binding and subsequent complex formation including enzymology.

Detection of Lectin Clustering in Self‐Assembled, Glycan‐Functionalized Amphiphiles by Aggregation‐Induced Emission Luminophores

AbstractAmphiphilic glycan‐functionalized oligomers are derived by solid‐phase polymer synthesis and applied in both, self‐assembled micelles as well as giant unilamellar vesicles, as simplified models of the cell's glycocalyx. Additionally, an aggregation‐induced luminophore is introduced into the amphiphilic glycomacromolecules showing no fluorescence when the molecule is free in solution. Combining glycomacromolecules carrying a binding glycan motif and the luminophore with glycomacromolecules or other amphiphiles with no binding motifs and no luminophore in self‐assembled structures, micelles and vesicles exhibiting no or only very little fluorescence are obtained. Only upon clustering of the binding glycan motifs through interaction with a multivalent lectin receptor, an increase in fluorescence is observed. Thus, clustering events within these self‐assembled structures can be detected and localized.

Atropine-functionalized gold nanoparticles binding to muscarinic receptors after passage across the intestinal epithelium.

Gold nanoparticles have a high potential to be a treatment of diseases by their specific drug delivery properties and multivalent receptor stimulation. For the present project, spherical gold nanoparticles were synthesized and functionalized with the muscarinic receptor antagonist atropine (Au-MUDA-AT NPs). The diameter of the gold core could precisely be controlled by using different synthetic methods and reducing agents resulting in functionalized gold nanoparticles with diameters ranging from 8 to 16 nm. The ability to interact with intestinal muscarinic receptors is size-dependent. When using intestinal chloride secretion induced by the stable acetylcholine derivative, carbachol, as read-out, the strongest inhibition, i.e. the most efficient blockade of muscarinic receptors, was observed with 13 nm sized Au-MUDA-AT NPs. Functional experiments indicate that Au-MUDA-AT NPs with a diameter of 14 nm are able to pass the intestinal mucosa in a time-dependent manner after administration to the intestinal lumen. For example, luminally administered Au-MUDA-AT NPs inhibited contractions of the small intestinal longitudinal muscle layer induced by electrical stimulation of myenteric neurons. A similar inhibition of basolateral epithelial receptors was observed after luminal administration of Au-MUDA-AT NPs when using carbachol-induced chloride secretion across the intestinal epithelium as a test system. Thus, Au-MUDA-AT NPs might be a therapeutic tool for the modulation of intestinal secretion and motility after oral application in the future.

"Willow Branch" DNA Self-Assembly for Cancer Dual-Target and Proliferation Inhibition.

DNA nanotechnology is beginning to yield unique advantages in the area of drug delivery. For the dual-targeting and proliferation suppression of cancer cells, a "willow branch" DNA assembly based on rolling circle amplification (RCA) was built. Three single-stranded DNAs, including antibody modified cDNAs, aptamer cDNAs, and simple cDNAs, were employed in the DNA self-assembly, along with the RCA scaffolds (every 63 bases is a repeat unit). "Willow branch" DNA (WB DNA) assembly successfully linked multiple antibodies and aptamers together to achieve dual targeting of cancer cells. Binding of CD44 antibodies and S2.2 aptamers to receptors on the cell membrane inhibits both pathways, β-catenin signaling and nuclear factor-kappa B-specific transcription activity, through feedback regulation. Results demonstrated that WB DNA assembly could effectively exert multivalency clustering cell-surface receptors, modulating signal pathways and inhibiting proliferation. This study proposes a new approach for cancer dual-target and proliferation inhibition by clustering multivalent receptors.

Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes dissociation of the S1 domains and exposure of the fusion machinery, although the molecular details of this process have yet to be observed. We report the development of bottom-up coarse-grained (CG) models consistent with cryo-electron tomography data, and the use of CG molecular dynamics simulations to investigate viral binding and S2 core exposure. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces in a manner distinct from binding between soluble proteins, which processively induces S1 dissociation. We also simulate possible variant behavior using perturbed CG models, and find that ACE2-induced S1 dissociation is primarily sensitive to conformational state populations and the extent of S1/S2 cleavage, rather than ACE2 binding affinity. These simulations reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.

Engineering non-antibody human proteins as efficient scaffolds for selective, receptor-targeted drug delivery

Self-assembling non-immunoglobulin scaffold proteins are a promising class of nanoscale carriers for drug delivery and interesting alternatives to antibody-based carriers that are not sufficiently efficient in systemic administration. To exploit their potentialities in clinics, protein scaffolds need to be further tailored to confer appropriate targeting and to overcome their potential immunogenicity, short half-life in plasma and proteolytic degradation. We have here engineered three human scaffold proteins as drug carrier nanoparticles to target the cytokine receptor CXCR4, a tumoral cell surface marker of high clinical relevance. The capability of these scaffolds for the selective delivery of Monomethyl auristatin E has been comparatively evaluated in a disseminated mouse model of human, CXCR4+ acute myeloid leukemia. Monomethyl auristatin E is an ultra-potent anti-mitotic drug used against a range of hematological neoplasias, which because of its high toxicity is not currently administered as a free drug but as payload in antibody-drug conjugates. The protein nanoconjugates generated here offer a collective strength of simple manufacturing process, high proteolytic and structural stability and multivalent ligand receptor interactions that result in a highly efficient and selective delivery of the payload drug and in a potent anticancer effect. The approach shown here stresses this class of human scaffold proteins as promising alternatives to antibodies for targeted drug delivery in the rapidly evolving drug development landscape.

A general model of multivalent binding with ligands of heterotypic subunits and multiple surface receptors

Multivalent cell surface receptor binding is a ubiquitous biological phenomenon with functional and therapeutic significance. Predicting the amount of ligand binding for a cell remains an important question in computational biology as it can provide great insight into cell-to-cell communication and rational drug design toward specific targets. In this study, we extend a mechanistic, two-step multivalent binding model. This model predicts the behavior of a mixture of different multivalent ligand complexes binding to cells expressing various types of receptors. It accounts for the combinatorially large number of interactions between multiple ligands and receptors, optionally allowing a mixture of complexes with different valencies and complexes that contain heterogeneous ligand units. We derive the macroscopic predictions and demonstrate how this model enables large-scale predictions on mixture binding and the binding space of a ligand. This model thus provides an elegant and computationally efficient framework for analyzing multivalent binding.

Cucurbit[7]urils induced bimetallic nanoparticles network for ultra-sensitive detection of Caspase-3 based on surface plasmon resonance

A novel signal amplification method for surface plasmon resonance (SPR) was developed to detect Caspase-3 based on cucurbit[7]urils (CB[7]) and bimetallic nanonetwork. Three-dimensional (3D) bimetallic nanonetwork was formed through CB[7]-integrated self-assembly of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) (AuNPs-CB[7]-AgNPs) by one-pot synthesis method. Caspase-3 was utilized as a model protein and peptide EEAAADEVDFKKAAAC was used as the substrate. In the presence of Caspase-3, peptide was hydrolyzed and exposed phenylalanine at the N-terminus, which could bind to CB[7] through host-guest interaction. The multivalent receptors CB[7] on bimetallic nanonetwork provide a simple strategy to increase the binding affinities between 3D bimetallic nanonetwork and peptide. Thus, AuNPs-CB[7]-AgNPs could attach to the gold chip with ease. Remarkably, due to the strong optical properties and high refractive index of 3D bimetallic nanonetwork, it has a higher SPR signal enhancement than that of only AuNPs or AgNPs. Therefore, a highly sensitive Caspase-3 sensing strategy could be achieved with a low detection limit of 5.39 pg mL−1, providing a new and facile SPR signal amplification method for detection of cell apoptosis.

Control of Superselectivity by Crowding in Three-Dimensional Hosts.

Motivated by the fine compositional control observed in membraneless droplet organelles in cells, we investigate how a sharp binding-unbinding transition can occur between multivalent client molecules and receptors embedded in a porous three-dimensional structure. In contrast to similar superselective binding previously observed at surfaces, we have identified that a key effect in a three-dimensional environment is that the presence of inert crowding agents can significantly enhance or even introduce superselectivity. In essence, molecular crowding initially suppresses binding via an entropic penalty, but the clients can then more easily form many bonds simultaneously. We demonstrate the robustness of the superselective behavior with respect to client valency, linker length, and binding interactions in MonteCarlo simulations of an archetypal lattice polymer model.

Abstract 518: Agonistic Death Receptor 5 (DR5) IgM antibody IGM-8444 induces tumor cell apoptosis in vitro and in vivo and has a favorable in vitro safety profile

Abstract Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that binds TNF-related apoptosis inducing ligand (TRAIL) to induce receptor trimerization and activate extrinsic apoptotic pathways. DR5 is expressed on a broad range of solid tumors as well as leukemias and lymphomas and has been targeted therapeutically with DR5 IgG antibodies and recombinant TRAIL agonists. However, agonistic IgG antibodies and TRAIL molecules targeting DR5 have been largely unsuccessful in clinical trials either in monotherapy or combination studies with chemotherapy due to insufficient receptor clustering to induce apoptotic pathways and/or due to very short half-life. Therefore, several new approaches have been taken to develop multivalent receptor DR5 agonists to enhance tumor cell apoptosis. We have taken the approach to efficiently cluster DR5 with multivalent IgM antibodies that contain 10 to 12 binding sites to DR5. First, we converted five agonistic IgG antibodies into the IgM antibody format and evaluated them using in vitro cell killing assays with colorectal cancer cell line Colo205 and compared them to IgG antibodies with the same binding domains. In all cases, we found the DR5 IgM antibodies significantly enhanced cellular cytotoxicity, resulting in at least 5,000 fold increased potency compared to IgGs. We have further developed a pentameric IgM antibody, IGM-8444, which has 10 binding sites specific for DR5 and efficiently triggers tumor cell apoptosis. In vitro tumor cell cytotoxicity screening identified IGM-8444 sensitive cell lines across 17 solid and hematologic cancer indications. IGM-8444 was efficacious as a monotherapy in both CDX and PDX preclinical mouse tumor xenograft models with complete and durable tumor regressions observed in a gastric PDX that expressed moderate levels of DR5 by immunohistochemistry. The combination of IGM-8444 with chemotherapeutic agents enhanced the cytotoxicity in several IGM-8444 resistant cell lines in vitro. IGM-8444 also combined well with standard-of-care therapies (e.g. irinotecan) in vivo, leading to enhanced efficacy. A significant challenge to the clinical development of multivalent DR5 agonists has been on-target liver toxicity. However, IGM-8444 had a favorable in vitro safety profile, with little/no in vitro cytotoxicity observed using primary human hepatocytes at concentrations >10,000 fold above the cytotoxicity EC50 observed in Colo205 cytotoxicity assays. Taken together, these data support the clinical development of IGM-8444 for the potential treatment of both solid and hematologic malignancies and an IND is projected to be filed in 2020. Citation Format: Beatrice Wang, Tasnim Kothambawala, Ling Wang, Bing Shan, Hai Lu, Ramesh Baliga, Marvin Peterson, Angus Sinclair, Bruce Keyt. Agonistic Death Receptor 5 (DR5) IgM antibody IGM-8444 induces tumor cell apoptosis in vitro and in vivo and has a favorable in vitro safety profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 518.

Template-Controlled Synthesis of Polyimidazolium Salts by Multiple [2+2] Cycloaddition Reactions.

The tetrakisimidazolium salt H4‐2(Br)4, featuring a central benzene linker and 1,2,4,5‐(nBu‐imidazolium‐Ph‐CH=CH‐) substituents reacts with Ag2O in the presence of AgBF4 to yield the tetranuclear, oktakis‐NHC assembly [3](BF4)4. Cation [3]4+ features four pairs of olefins from the two tetrakis‐NHC ligands perfectly arranged for a subsequent [2+2] cycloaddition. Irradiation of [3](BF4)4 with a high pressure Hg lamp connects the two tetra‐NHC ligands through four cyclobutane linkers to give compound [4](BF4)4. Removal of the template metals yields the novel oktakisimidazolium salt H8‐5(BF4)8. The tetrakisimidazolium salt H4‐2(BF4)4 and the oktakisimidazolium salt H8‐5(BF4)8 have been used as multivalent anion receptors and their anion binding properties towards six different anions have been compared.

Antiviral Activity of Chicken Cathelicidin B1 Against Influenza A Virus.

Cathelicidins (CATHs) are host defense peptides (HDPs) that play an important role in the innate immune response against infections. Although multiple functions of cathelicidins have been described, including direct antimicrobial activity and several immunomodulatory effects on the host, relatively little is known about their antiviral activity. Therefore, in vitro antiviral activity of chicken cathelicidins and the underlying mechanism was investigated in this study against different influenza A virus (IAV) strains. Our results show that chicken CATH-B1 has broad anti-IAV activity compared to other cathelicidins (CATH-1, -2, -3, LL-37, PMAP-23, and K9CATH) with an inhibition of viral infection up to 80% against three tested IAV strains (H1N1, H3N1, and H5N1). In agreement herewith, CATH-B1 affected virus-induced inflammatory cytokines expression (IFN-β, IL-1β, IL-6, and IL-8). Incubation of cells with CATH-B1 prior to or after their inoculation with virus did not reduce viral infection indicating that direct interaction of virus with the peptide was required for CATH-B1’s antiviral activity. Experiments using combined size exclusion and affinity-based separation of virus and peptide also indicated that CATH-B1 bound to viral particles. In addition, using electron microscopy, no morphological change of the virus itself was seen upon incubation with CATH-B1 but large aggregates of CATH-B1 and viral particles were observed, indicating that aggregation might be the mechanism of action reducing IAV infectivity. Neuraminidase (NA) activity assays using monovalent or multivalent substrates, indicated that CATH-B1 did not affect NA activity per se, but negatively affected the ability of virus particles to interact with multivalent receptors, presumably by interfering with hemagglutinin activity. In conclusion, our results show CATH-B1 has good antiviral activity against IAV by binding to the viral particle and thereby blocking viral entry.

TOLs Function as Ubiquitin Receptors in the Early Steps of the ESCRT Pathway in Higher Plants

Protein abundance and localization at the plasma membrane (PM) shapes plant development and mediates adaptation to changing environmental conditions. It is regulated by ubiquitination, a post-translational modification crucial for the proper sorting of endocytosed PM proteins to the vacuole for subsequent degradation. To understand the significance and the variety of roles played by this reversible modification, the function of ubiquitin receptors, which translate the ubiquitin signature into a cellular response, needs to be elucidated. In this study, we show that TOL (TOM1-like) proteins function in plants as multivalent ubiquitin receptors, governing ubiquitinated cargo delivery to the vacuole via the conserved Endosomal Sorting Complex Required for Transport (ESCRT) pathway. TOL2 and TOL6 interact with components of the ESCRT machinery and bind to K63-linked ubiquitin via two tandemly arranged conserved ubiquitin-binding domains. Mutation of these domains results not only in a loss of ubiquitin binding but also altered localization, abolishing TOL6 ubiquitin receptor activity. Function and localization of TOL6 is itself regulated by ubiquitination, whereby TOL6 ubiquitination potentially modulates degradation of PM-localized cargoes, assisting in the fine-tuning of the delicate interplay between protein recycling and downregulation. Taken together, our findings demonstrate the function and regulation of a ubiquitin receptor that mediates vacuolar degradation of PM proteins in higher plants.

Synthesis of a first-generation l-rhamnose dendron

During the last few years, various strategies for cancer therapy have emerged where l-rhamnose interacts with either an anti-rhamnose antibody or a hypothetically over-expressed l-rhamnose-binding lectin. These studies have led to promising results in cancer immunotherapy and the design of selective cytotoxic rhamnosides, respectively. We have synthesised an l-rhamnose dendron designed to amplify the interaction between l-rhamnose and multivalent receptors. The dendron was prepared in a very good 27% yield over 8 steps. It features an azide bearing side chain making it a versatile building block for the construction of innovative glycoconjugates relying on the peculiar properties of l-rhamnose for cancer treatment.

Spatial Requirements for T-Cell Receptor Triggering Probed via Functionalized DNA Origami Platforms

Controlling the spatial organization of ligands on cell surfaces is emerging as a powerful tool to assess the relevance of multivalent binding, receptor clustering and molecular cooperativity for cell signaling. A mechanistic understanding of such processes would without doubt accelerate rational approaches in modern pharmacology and cell-based therapies.Here, we have designed a biomimetic interface based on lipid bilayer-anchored DNA origami nanostructures as a tool to study the spatial requirements for receptor-mediated signaling in cell-cell contacts.

Multivalent Interactions of Polyamide Based Sequence‐Controlled Glycomacromolecules with Concanavalin A

Binding of mannose presenting macromolecules to the protein receptor concanavalin A (ConA) is investigated by means of single-molecule atomic force spectroscopy (SMFS) in combination with dynamic light scattering and molecular modeling. Oligomeric (Mw ≈ 1.5-2.5 kDa) and polymeric (Mw ≈ 22-30 kDa) glycomacromolecules with controlled number and positioning of mannose units along the scaffolds accessible by combining solid phase synthesis and thiol-ene coupling are used as model systems to assess the molecular mechanisms that contribute to multivalent ConA-mannose complexes. SMFS measurements show increasing dissociation force from monovalent (≈57 pN) to pentavalent oligomers (≈75 pN) suggesting subsite binding to ConA. Polymeric glycomacromolecules with larger hydrodynamic diameters compared to the binding site spacing of ConA exhibit larger dissociation forces (≈80 pN), indicating simultaneous dissociation from multiple ConA binding sites. Nevertheless, although simultaneous dissociation of multiple ligands could be expected for such multivalent systems, predominantly single dissociation events are observed. This is rationalized by strong coiling of the macromolecules' polyamide backbone due to intramolecular hydrogen bonding hindering unfolding of the coil. Therefore, this study shows that the design of glycopolymers for multivalent receptor binding and clustering must consider 3D structure and intramolecular interactions of the scaffold.

A novel discovery, maturation, and assay integration approach for the development of ruggedized multi-valent capture receptors exemplified against the chikungunya virus E2 protein

This work reports on the development of synthetic, thermally stable receptors using a novel discovery and multi-valent maturation strategy exemplified against the Chikungunya Virus (CHIKV) E2 surface protein capable of straightforward bioassay integration. An in situ ‘click’ screen of a propargylated folded protein against a comprehensive library of peptide macrocycles with 5-mer variable regions produced four candidate binders with nanomolar affinities. With the guidance of in silico experiments, the individual macrocycles were matured to produce multi-valent cooperative constructs, resulting in successful binding of CHIKV E2 in up to 50% human serum and an over 200-fold improvement in affinity performance compared to the mono-valent macrocycles. The top performing multi-valent construct also exhibits an insignificant loss in capture performance after being heated up to 90 °C for 1 h. The overall methodology provides a rational approach to the general targeting of folded proteins, providing the ability to rapidly develop drop-in biological assay antibody replacements and potentially “polyclonal” mixtures of reagents.

Multivalent Crown Ether Receptors Enable Allosteric Regulation of Anion Exchange in an Fe4L6 Tetrahedron

AbstractWe report a strategy for regulating the rate of internally bound anion exchange within an Fe4L6 metal–organic tetrahedron through external coordination of tripodal tris(alkylammonium) cations. The cage features three flexible 18‐crown‐6 receptors at each of its FeII vertices, facilitating strong tritopic interactions with tris(ammonium) cations to “cap” the vertices of the tetrahedron. This capping mechanism restricts the flexibility of the cage framework, thereby reducing the rate of anion exchange within its central cavity by 20‐fold. Thus, we demonstrate the first use of an externally bound multivalent effector to allosterically control internal guest binding in a molecular cage.