Respiratory syncytial virus-approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors.

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus-approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy.

Chemically Synthetic Membrane Receptors Establish Cells with Artificial Sense-and-Respond Signaling Pathways.

Chemically synthetic receptors that establish cells a new sense-and-respond capability to interact with outer worlds are highly desired, but rarely reported. In this work, we develop a membrane-anchored synthetic receptor (Ts-pHLIP-Pr) using DNA and peptide as the building block to equip cells with artificial signaling pathways. Upon sensing external pH stimuli, the Pr module can be translocated across the cell membrane via the conformation switch of pHLIP, enabling membrane-proximal recruitment of specific proteins to trigger downstream signaling cascades. Our experimental results demonstrate the capability of Ts-pHLIP-Pr for regulating PKCε-related signaling events upon responding to external pH reduction. With a modular feature, this receptor can be extended to elicit T cell activation through low-pH environment-induced directional movement of cytoplasmic ZAP70. Our work is expected to offer a new paradigm for intelligent synthetic biology and customized cell engineering.

Semisynthesis of Aminomethyl-Insulin: An Atom-Economic Strategy to Increase the Affinity and Selectivity of a Protein for Recognition by a Synthetic Receptor.

Advancements in the molecular recognition of insulin by nonantibody-based means would facilitate the development of methodology for the continuous detection of insulin for the management of diabetes mellitus. Herein, we report a novel insulin derivative that binds to the synthetic receptor cucurbit[7]uril (Q7) at a single site and with high nanomolar affinity. The insulin derivative was prepared by a four-step protein semisynthetic method to present a 4-aminomethyl group on the side chain of the PheB1 position. The resulting aminomethyl insulin binds to Q7 with an equilibrium dissociation constant value of 99 nM in neutral phosphate buffer, as determined by isothermal titration calorimetry. This 6.8-fold enhancement in affinity versus native insulin was gained by an atom-economical modification (-CH2NH2). To the best of our knowledge, this is the highest reported binding affinity for an insulin derivative by a synthetic receptor. This strategy for engineering protein affinity tags induces minimal change to the protein structure while increasing affinity and selectivity for a synthetic receptor.

Modulating the Influenza A Virus-Target Membrane Fusion Interface With Synthetic DNA-Lipid Receptors.

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell-surface glycans are highly heterogeneous in length and glycosylation density, which leads to variations in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion or the low-pH-triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

The challenges of glycan recognition with natural and artificial receptors.

Glycans - simple or complex carbohydrates - play key roles as recognition determinants and modulators of numerous physiological and pathological processes. Thus, many biotechnological, diagnostic and therapeutic opportunities abound for molecular recognition entities that can bind glycans with high selectivity and affinity. This review begins with an overview of the current biologically and synthetically derived glycan-binding scaffolds that include antibodies, lectins, aptamers and boronic acid-based entities. It is followed by a more detailed discussion on various aspects of their generation, structure and recognition properties. It serves as the basis for highlighting recent key developments and technical challenges that must be overcome in order to fully deal with the specific recognition of a highly diverse and complex range of glycan structures.

Multisite Binding of Drugs and Natural Products in an Entropically Favorable, Heteroleptic Receptor.

The cavities of artificial receptors are defined by how their components fit together. The encapsulation of specific molecules can thus be engineered by considering geometric principles; however, intermolecular interactions and steric fit scale with receptor size, such that the ability to bind multiple guests from a specific class of compounds remains a current challenge. By employing metal-organic self-assembly, we have prepared a triangular prism from two different ligands that is capable of binding more than 20 different natural products, drugs, and steroid derivatives within its prolate cavity. Encapsulation inflates the host, enhancing its ability to bind other guests in peripheral pockets and thus enabling our system to bind combinations of different drug and natural product cargoes in different locations simultaneously. This new mode of entropically favorable self-assembly thus enables central encapsulation to amplify guest-binding events around the periphery of an artificial receptor.

Achieving High Affinity and Selectivity for Asymmetric Dimethylarginine by Putting a Lid on a Box.

The methylation states of Lys and Arg represent a particularly challenging set of targets to distinguish selectively in water using synthetic receptors. To date, trimethyllysine (Kme3) is the only post translational modification (PTM) of the eight possible methylation states of Lys and Arg that can be recognized selectively. Here, we report the first synthetic receptor capable of selectively recognizing asymmetric dimethylarginine (Rme2a). This was achieved by using a biased dynamic combinatorial chemistry (DCC) library to generate a receptor mimicking the 5-sided box-like shape of Rme2 reader proteins, a feature that has been hypothesized to impart selectivity. Additionally, we synthesized a thioether-linked analogue of the resulting receptor to provide a novel scaffold with maintained selectivity but greater stability. This work introduces strategies that can be applied towards achieving selectivity based on subtle differences in hydrophilic guests in aqueous solutions.

Synthetic Receptors for the Recognition and Discrimination of Post-Translationally Methylated Lysines.

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology, including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side-chain methylation of lysine. For the analysis and discrimination of differently methylated lysines, antibodies are widely used, although methylated peptide and protein targets are known to be particularly difficult to differentiate by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation procedure and is not suited for working in cells. The desire to overcome the above-mentioned challenges has promoted the development of synthetic receptor molecules that recognise and bind methyllysines. Such "artificial antibodies" are of interest for a number of applications, for example, as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines, including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbiturils and acyclic receptors.

Selective Binding of Folic Acid and Derivatives by Imprinted Nanoparticle Receptors in Water.

Folate receptors are overexpressed on cancer cells and frequently used for targeted delivery. Creation of synthetic receptors to bind folic acid and its analogues in water, however, is challenging because of its complex hydrogen-bonding patterns and competition for hydrogen bonds from the solvent. Micellar imprinting within cross-linkable surfactants circumvented these problems because the nonpolar micellar environment strengthened the hydrogen bonds between the amide group in the surfactant and the template molecule. Incorporation of polymerizable thiouronium functional monomers further enhanced the binding through hydrogen-bond-reinforced ion pairs with the glutamate moiety of the template. The resulting imprinted micelles were able to bind folate and their analogues with submicromolar affinity and distinguish small changes in the hydrogen-bonding patterns as well as the number/position of carboxylic acids. The binding constant obtained was 2-3 orders of magnitude higher than those reported for small-molecule synthetic receptors. Our binding study also revealed interesting details in the binding. For example, the relative contributions of different segments of the molecule to the binding followed the order of carboxylates > pyrimidine ring > pyrazine ring.

A Biomimetic Synthetic Receptor Selectively Recognising Fucose in Water.

Carbohydrate recognition in water by biomimetic receptors is an attractive, but very challenging goal. Despite advances achieved in glucose recognition, little or no success has been obtained in the recognition of other saccharidic epitopes of paramount importance in biological processes. Herein, the unprecedented recognition of fucose in water by an artificial receptor that shows affinities closely comparable to those of several lectins is reported. The receptor has been constructed by assembling a hydrogen-bonding element (carbazole), a hydrophobic aromatic moiety (anthracene), and a water-solubilising function (phosphonate) into a macrocyclic structure to provide the appropriate binding geometry. The described receptor binds fucose with sub-millimolar affinity in water at physiological pH; this shows that enthalpic binding can be ascribed to hydrogen bonding to saccharidic hydroxy groups and to CH-π interactions between the sugar backbone and aromatic moieties. Experimental NOE contacts coupled to conformational search calculations return a picture of a binding site in which fucose assumes a staggered orientation reminiscent of that shown by fucose when bound to the Ralstonia solanacearum lectin (RSL).

Use of Membrane Potential to Achieve Transmembrane Modification with an Artificial Receptor.

We developed a strategy to modify cell membranes with an artificial transmembrane receptor. Coulomb force on the receptor, caused by the membrane potential, was used to achieve membrane penetration. A hydrophobically modified cationic peptide was used as a membrane potential sensitive region that was connected to biotin through a transmembrane oligoethylene glycol (OEG) chain. This artificial receptor gradually disappeared from the cell membrane via penetration despite the presence of a hydrophilic OEG chain. However, when the receptor was bound to streptavidin (SA), it remained on the cell membrane because of the large and hydrophilic nature of SA.

Development of a synthetic receptor protein for sensing inflammatory mediators interferon-γ and tumor necrosis factor-α.

Intestinal inflammation has been implicated in a number of diseases, including diabetes, Crohn's disease, and irritable bowel syndrome. Important components of inflammation are interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are elevated both on the luminal and submucosal sides of the intestinal epithelial barrier in several diseases. Here, we developed a novel Escherichia coli based detection system for IFN-γ and TNF-α comprised of a chimeric protein and a simple signal transduction construct, which could be deployed on the luminal side of the intestine. OmpA of E. coli was engineered to detect IFN-γ or TNF-α through the replacement of extracellular loops with peptide fragments from OprF of P. aeruginosa. OmpA/OprF chimeras were developed, capable of binding IFN-γ or TNF-α. The specific peptide fragments that bind IFN-γ were identified. IFN-γ or TNF-α binding the OmpA/OprF chimera induced the pspA promoter, driving ß-galactosidase production. The OmpA/OprF chimera had a detection limit of 300 pM for IFN-γ and 150 pM for TNF-α. This work will further the development of bacteria based therapeutics for the treatment of inflammatory diseases of the gut.

Integrating sampling techniques and inverse virtual screening: toward the discovery of artificial peptide-based receptors for ligands.

A novel heuristic using an iterative select-and-purge strategy is proposed. It combines statistical techniques for sampling and classification by rigid molecular docking through an inverse virtual screening scheme. This approach aims to the de novo discovery of short peptides that may act as docking receptors for small target molecules when there are no data available about known association complexes between them. The algorithm performs an unbiased stochastic exploration of the sample space, acting as a binary classifier when analyzing the entire peptides population. It uses a novel and effective criterion for weighting the likelihood of a given peptide to form an association complex with a particular ligand molecule based on amino acid sequences. The exploratory analysis relies on chemical information of peptides composition, sequence patterns, and association free energies (docking scores) in order to converge to those peptides forming the association complexes with higher affinities. Statistical estimations support these results providing an association probability by improving predictions accuracy even in cases where only a fraction of all possible combinations are sampled. False positives/false negatives ratio was also improved with this method. A simple rigid-body docking approach together with the proper information about amino acid sequences was used. The methodology was applied in a retrospective docking study to all 8000 possible tripeptide combinations using the 20 natural amino acids, screened against a training set of 77 different ligands with diverse functional groups. Afterward, all tripeptides were screened against a test set of 82 ligands, also containing different functional groups. Results show that our integrated methodology is capable of finding a representative group of the top-scoring tripeptides. The associated probability of identifying the best receptor or a group of the top-ranked receptors is more than double and about 10 times higher, respectively, when compared to classical random sampling methods.

Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives.

The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc-ß-OMe with Ka ≈20,000 m(-1), whereas the other one binds an O-GlcNAcylated peptide with Ka ≈70,000 m(-1). These values substantially exceed those usually measured for GlcNAc-binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl-peptide complex can be explained by extra-cavity interactions, raising the possibility of a family of complementary receptors for O-GlcNAc in different contexts.

A Synthetic Supramolecular Receptor for the Hydrosulfide Anion.

Hydrogen sulfide (H2 S) has emerged as a crucial biomolecule in physiology and cellular signaling. Key challenges associated with developing new chemical tools for understanding the biological roles of H2 S include developing platforms that enable reversible binding of this important biomolecule. The first synthetic small molecule receptor for the hydrosulfide anion, HS(-) , using only reversible, hydrogen-bonding interactions in a series of bis(ethynylaniline) derivatives, is reported. Binding constants of up to 90 300±8700 m(-1) were obtained in MeCN. The fundamental science of reversible sulfide binding, in this case featuring a key CH⋅⋅⋅S hydrogen bond, will expand the possibility for discovery of sulfide protein targets and molecular recognition agents.

Synthetic sialylglycopolymer receptor for virus detection using cantilever-based sensors.

We describe the rapid, label-free detection of Influenza A viruses using a cantilever transducer modified with a synthetic sialylglycopolymer receptor layer. Surface stresses induced by viruses binding to the receptor layer were used as the analytical signal. The synthetic sialylglycopolymer receptor layer can be used in nanoscale strain-gauge cantilever transducers for highly sensitive virus detection. Strain-gage transducers using such sensor layers exhibit long lifetimes, high sensitivities, and possible regeneration. Nanomechanical cantilever systems using optical detectors were used for the surface stress measurements. We demonstrated the positive, label-free detection of Influenza A at concentrations below 10(6) viruses per ml. In contrast to hemagglutination assays, cantilever sensors are label free, in situ, and rapid (less than 30 min), and they require minimal or nearly no sample preparation.

Affinity enhancement by dendritic side chains in synthetic carbohydrate receptors.

Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted in binding substrates rather than blocking the cavity. Conformational degeneracy in the dendrimers resulted in effective preorganization despite the flexibility of the system. Strong binding was observed to glucosammonium ions in water, with Ka values up to 7000 M(-1) . Affinities for uncharged substrates (glucose and N-acetylglucosamine) were also enhanced, despite competition from solvent and the absence of electrostatic interactions.

Synthetic receptor scaffolds significantly affect the efficiency of cell fate signals.

Mimicry of receptor functions by designing synthetic receptors would be one of the recently hot research trends in cell engineering. While several types of synthetic receptors have been designed to induce desired cell fates in response to external stimuli, little is known about which receptor type signals more efficiently for inducing a certain cell fate. In this study, we compared the performance of three types of synthetic receptor scaffolds, i.e. myristoylated, cytosolic, and transmembrane types that signal through JAK-dependent phosphorylation of tyrosine motifs to transduce growth signaling. As a result, the phosphorylation levels of JAK and subsequent downstream signaling molecules were significantly maintained in the cytosolic type receptors, leading to more efficient cell growth than the other types. In contrast, the phosphorylation levels of JAK decreased in a motif-dependent manner in the transmembrane type receptors. Although various studies on receptor engineering based on domain or motif engineering have been reported, to our knowledge this study is the first to demonstrate that synthetic receptor scaffolds significantly affect the efficiency of cell fate signals. These findings are important for both receptor biology and receptor engineering, providing guidelines for rationally designing synthetic receptors that can transduce as efficient signaling as possible.

Artificial Tactile Receptor System for Sensitive Pressure-Neural Spike Conversion.

An artificial tactile receptor is crucial for e-skin in next-generation robots, mimicking the mechanical sensing, signal encoding, and preprocessing functionalities of human skin. In the neural network, pressure signals are encoded in spike patterns and efficiently transmitted, exhibiting low power consumption and robust tolerance for bit error rates. Here, we introduce a highly sensitive artificial tactile receptor system integrating a pressure sensor, axon-hillock circuit, and neurotransmitter release device to achieve pressure signal coding with patterned spikes and controlled neurotransmitter release. Owing to the heightened sensitivity of the axon-hillock circuit to pressure-mediated current signals, the artificial tactile receptor achieves a detection limit of 10 Pa that surpasses the human tactile receptors, with a wide response range from 10 to 5 × 105 Pa. Benefiting from the appreciable pressure-responsive performance, the potential application of an artificial tactile receptor in robotic tactile perception has been demonstrated, encompassing tasks such as finger touch and human pulse detection.

The Hamilton Receptor in Supramolecular Polymer Sciences.

Supramolecular polymers are polymeric materials of monomeric fragments, held jointly by reversible and directional non-covalent interactions such as multiple hydrogen-bonding, charge transfer effects, host-guest interactions, metal coordination, and aromatic stacking. This review article on the Hamilton-based supramolecular polymers aims to shed light on the molecular recognition achievements by the Hamilton-based polymeric systems, evaluate Hamilton receptor's future prospects, and capitalize its potential applications in supramolecular chemistry. To the best of our knowledge, this is the first elaborative and sole manuscript in which polymeric Hamilton receptors are being exposed in detail. The first portion of this manuscript is related to the importance and urgency of polymers along with the historic background of Hamilton receptors. The middle section discloses the potential applications of Hamilton-type receptors in various fields, e.g., dendrimers, mechanically polymeric rotaxanes, and self-assemblies. The final section of the manuscript discloses the future aspects and the importance of novel polymer-based Hamilton-type receptors in the modern era. We believe that this first review in this emerging yet immature field will be useful to inspire scientists around the world to find the unseen future prospects, thereby boosting the field related to this valued artificial receptor in the province of supramolecular chemistry and also in other domains of scientific fields and technology, as well.